NINDS GENSAT BAC TRANSGENIC PROJECT Supported by NINDS Contract # N01-NS

Size: px
Start display at page:

Download "NINDS GENSAT BAC TRANSGENIC PROJECT Supported by NINDS Contract # N01-NS"

Transcription

1 NINDS GENSAT BAC TRANSGENIC PROJECT Supported by NINDS Contract # N01-NS The Rockefeller University 1. BAC Transgene Construction Shiaoching Gong The plasmid, pld53.sc2, used to generate EGFP lines is a derivative of pld53. PLD53 was digested with BamHI and SacI to get rid of the tetar and orit origin and it was replaced by a NotI-SalI-SpeI adaptor. A 1.1 kb EGFP-PA was cloned into NotI and SalI sites, and a AscI-NotI-SwaI-SmaI multiple cloning site was then subcloned into the NotI site. The original NotI site was knocked out hereafter. This shuttle vector (pld53.sc2) was digested with Asc/Sma1 and purified by running on a 1% low melting agarose gel. A bp A Box fragment used for the homologous recombination was PCR amplified, digested with AscI and cloned into this predigested shuttle vector as described below. The plasmid, PLD53.SC.Cre, used to generate the Cre lines is also a derivative of pld53. PLD53 was digested with BamHI and SacI to get rid of the tetar and orit origin and replaced by AscI-NotI-SwaI-SmaI-SalI adaptor. A 1.3 Kb fragment containing Cre-Intron-Cre and HSFVTK polya was cloned into SmaI/SalI sites. The steps for A box cloning are the same as above. Transform the pld53.sc2 or pld53.sc.cre shuttle vector into PIR1 or PIR2 E. coli cells (Invitrogen, C or C ), transform psvreca plasmid into DH5a E. coli cells (Invitrogen, cat no ). Preparation of DNA for pld53.sc2 and PSV-RecA 1. To grow pld53.sc2, which possess an ampicillin resistant gene, pick a single colony of pld53.sc2 transformed Pir1 bacteria from the plate and inoculate 3 ml of Luria Broth medium supplemented with ampicillin (50

2 ug/ml). Incubate at 37 0 C for 8 hours. Transfer this culture into 500 ml of Luria Broth medium supplemented with ampicillin (50 ug/ml) and incubate for h at 37 0 C with shaking at 300 rpm, 2. To grow psv1.reca, which possess a tetracycline resistant gene, pick a single colony of psv1.reca transformed DH5a bacteria from the plate and inoculate 3 ml of Luria Broth medium supplemented with Tet (10 μg/ml) Incubate at 30 0 C for 8 hours. Transfer this culture into 500 ml of Luria Broth medium supplemented with Tet (10 μg/ml) and incubate for h at 30 0 C with shaking at 300 rpm, 3. Spin down the bacteria at 4552 g for 30 mins at 4 0 C (J6-MI Beckman- Coulter centrifuge, JS-4.2 rotor). 4. Discard supernatant and resuspend bacterial pellet in 30 ml of P1 buffer (Qiagen, Cat no ). 5. Add 30 ml of P2 buffer (Qiagen, Cat no ), mix gently and incubate at room temperature for 5 min. 6. Add 30 ml of ice-cold P3 buffer (Qiagen, Cat no ), mix gently and incubate on ice for 20 min. 7. Spin at g for 30 mins at 4 0 C (J-25I Beckman Avanti centrifuge, JLA rotor). 8. Add 63 ml of isopropanol to supernatant, mix and collect DNA by centrifugation Spin at g for 30 mins at 4 0 C (J-25I Beckman Avanti centrifuge, JLA rotor). Discard the supernatant and wash the pellet with 70% EtOH, dry at RT for 5-10 minutes and resuspend in 5 ml 1x TE. 9. Extract the DNA with an equal volume of phenol/chloroform (Sigma, Cat no. P4557) and spin down for 5 minutes. 10. Transfer the aqueous phase into another tube. Add 2 volumes of EtOH, mix and collect DNA by centrifugation at g at 4 0 C for 30 minutes. 11. Discard the supernatant and wash the pellet with 70% EtOH. Dry the pellet room temperature resuspend it in 400 μl of 1x TE. 12. Prepare Chroma Spin-400 Columns (Clontech, Cat no. K1323-2) by Inverting the CHROMA spin-400 columns (Clontech) several times to resuspend the gel matrix completely. 13. Grasp the break-away end and lift off the top cap. Place the end of the spin column into one of the 2-ml microcentrifuge tube. Hold them with a Falcon 14 ml polypropylene round-bottom tube (2059). 14. Centrifuge at 700 g for 5 mins at 4 0 C (J6-MI Beckman-Coulter centrifuge, JS-4.2 rotor). 15. Discard the 2-ml collection tube with the buffer. Place the spin column into the second 2-ml microcentrifuge tube. Hold them with a Falcon 14

3 ml polypropylene round-bottom tube (2059). Carefully and slowly apply the DNA sample from step 10 into center of the column. Do not allow any sample to flow along the inner wall of the column. 16. Centrifuge at 700 g for 5 mins at 4 0 C (J6-MI Beckman-Coulter centrifuge, JS-4.2 rotor). Save the DNA and check its concentration. Cloning of shuttle vectors 1. Prepare 100 ug (enough for 1000 ligation reactions) of the AscI/SwaI digested pld53.sc2 shuttle vector by incubation overnight in appropriate amounts of enzyme. Purify the digested vector, test the aliquot by ligation to determine background of undigested or single digested shuttle vector, redigest it until the background disappears. Aliquot and store this stock of predigested vector for use in A box cloning. 2. Prepare DNA from the selected BAC clone, resuspend the BAC DNA in 50 μl of 1x TE (10mM Tris, ph 8.0, 1 mm EDTA). PCR amplify a bp A box homology regions from 1 μl of the BAC DNA (5 primer incorporates AscI site and 3 primer does not incorporate any restriction sites). Precipitate the DNA from the PCR reaction, and resuspend it with 86 ul of H 2 O, digest the products overnight with 4 ul of AscI, purify digested fragments by QIAquick PCR purification kit (Qiagen, Cat no ). 3. Ligate the digested shuttle vector (100 ng) with each individual fragment (60-80 ng), transform into PIR1 or PIR2 cells and plate the transformed cells on ampicillin (30ug/ml) plates. Use 20 ul of PIR1 or PIR2 cells + 2 ul of ligation mixture. 4. Test individual colonies for correct insertion by PCR (using R6K gamma primer and 3 end primer of the A Box). Prepare DNA for each positive shuttle vector and confirm these clones with restriction enzymes (AscI/SmaI) by comparing the digestion pattern with the vector. Since this shuttle vector contains a R6kr DNA replication origin, which can only replicate in bacteria expressing the pir replication protein, use of PIR1 or PIR2 cells from Invitrogen is strongly recommended. During this step, the A box fragment should not contain an internal Asc I site. If so, you have to incorporate a Mlu I site at the 5 primer and digest your PCR product with Mlu I.

4 Preparation of the BAC shuttle vector 1. In a 50 ml Falcon tube, inoculate 15 ml of broth (7.5 ml of Luria Broth and 7.5 ml of Terrific Broth) supplemented with 30ug/ml of ampicillin with one colony. Incubate at 37 0 C overnight. 2. Harvest cells by centrifugation at 4552 g (J6-MI) for 20 minutes at 4 0 C. 3. Discard the supernatant and add 500 ul of P1 buffer (Qiagen, Cat no ) to resuspend the bacteria pellet. 4. Add 500 ul of P2 buffer (Qiagen, Cat no ) to lysate the bacteria, mix well and incubate at room temperature for 5 minutes. 5. Add 500 ul of ice-cold P3 buffer (Qiagen, Cat no ) to neutralize the lysate, mix well. 6. Pour the mixture into a 2.0 ml Eppendorf tube. 7. Centrifuge at g for 30 minutes. 8. Split volume between two (about 750ul each) 2.0 ml Phase Lock Gels tubes (Fisher, Cat no ) and extract with an equal volume of phenol/chloroform (Sigma, Cat no. P4557). Place tubes on an orbital mixer for 5 minutes. 9. Transfer the top layer of each tube into a set of 2.0 ml sterile Eppendorf tubes. 10. Fill each tube with 100% EtOH and mix well. 11. Centrifuge at g for 30 minutes. Wash the pellet with 70% EtOH. 12. Dry and resolve both pellets with 200 ul of 1x TE. 13. Prepare Chroma Spin-400 Columns (Clontech, Cat no. K1323-2). 14. Transfer the DNA into the center of the spin column. 15. Centrifuge at 700 g for 5 minutes. 16. Transfer the DNA (200 μl) into another 1.5 ml Eppendorf tube, add 100 μl of 7.5 M ammonium acetate and 750 ul of 100% EtOH. 17. Place tubes in 70 0 C freezer for 5 minutes and centrifuge at g for 30 minutes. Wash the pellet with 70% EtOH. 18. Resuspend the pellet with μl of TE. 19. Check the DNA concentration on an agarose gel. Preparation of BAC host cells by chemical transformation 1. Pick a single colony from the LB agar plate supplemented with chloramphenicol (20 ug/ml) and inoculate 5ml of Luria Broth medium supplemented with chloramphenicol (20 ug/ml). Grow the cells overnight at 37 0 C with shaking at 300 r.p.m. Dilute the overnight culture

5 1:100 with LB supplemented with chloramphenicol (20 ug/ml) and incubate for 3-4 hours at 37 0 C to an OD 600nm of Harvest the cells by centrifugation at 2560 g for 10 min at 4 0 C. 3. Discard the supernatant and resuspend the bacterial pellet in 5 ml of icecold 50mM CaCl2. Place on ice for 15 minutes and re-harvest as above. 4. Resuspend the pellet in 300 ul of ice-cold solution of 50 mm CaCl2 and 20% glycerol. Aliquot 100 ul into 3 tubes and save two of these butes at C. 5. Thaw an aliquot of psv1.reca DNA made in an earlier step and add 2-5 ul of this plasmid to 100 ul of the BAC host cells. Mix the tube gently and incubate for 30 min on ice. 6. Heat shock the bacteria by placing the tube in a 42 0 C water bath for 50 s. 7. Add 1.0 ml of SOC medium to the mixture, transfer into a 1.5 ml eppendorf tube and incubate for 1 h at 30 0 C for 1 hour with shaking at 225 rpm. 8. Spread either 5 ul or 100 ul of the transformed BAC cells onto two LB agar plate supplemented with chloramphenicol (20 ug/ml) and tetracyclin (10 μg/ml). Incubate overnight at 30 0 C. 9. Pick a single colony from the plate and inoculate 5 ml of Luria Broth medium supplemented with chloramphenicol (20 ug/ml) and tetracyclin (10 μg/ml). Incubate overnight at 30 0 C with shaking at 300 rpm. 10. Transfer 1 or 2 ml of overnight culture into 50 ml of LB supplemented chloramphenicol (20 ug/ml) and tetracyclin (10 μg/ml) in a 250 ml Corning centrifuge tube. Grow cells for 4-5 h with vigorous shaking at 30 0 C to an OD 600nm of Harvest cells by centrifugation at 2560 g at 4ºC for 10 min (J6-MI Beckman Coulter centrifuge, JS-4.2 rotor). 12. Resuspend the pellet in an equal volume of 10% cold glycerol and centrifuge at 2560 g for 10 min at 4 0 C J6-MI Beckman Coulter centrifuge, JS-4.2 rotor). 13. Repeat steps 11 and 12 one time. 14. Decant the supernatant as much as possible and gently resuspend the cells to a final volume of 200 ul with cold 10% glycerol. Glycerol solution can be difficult to decant. Make sure that your final dilution of cells is not greater than 200 ul. 15. Aliquot 40 ul into 5 Eppendorf tubes and store at C.

6 Transformation of BAC host competent cells via electroporation 1. Thaw 40 ul of psv1.reca transformed-bac competent cells on ice and mix with 2 ul of shuttle vector DNA (500 ng/ul). Place the mixture on ice for 1 min and transfer each sample to a cold 0.1 cm cuvette (BIO-RAD, Cat # ). Set BioRad Gene Pulser II apparatus at 25uF, the voltage to 1.8kV and pulse controller to 200Ω. 2. After electroporation, add 1.0 ml of SOC to each cuvette and transfer the cell suspension to a 15 ml tube and incubate at 30 0 C for 1.0 hour with shaking at 225 rpm. 3. Transfer sample into 5 ml of Luria Broth containing chloramphenicol (20 ug/ml) and tetracyclin (10 μg/ml) Tet (10 ug/ml), and ampicillin (50 ug/ml) and incubate at 30 0 C overnight. 4. Spread 20 ul and 100 ul of the overnight culture onto Luria Broth plates containing chloramphenicol (20 ug/ml) and ampicillin (50 ug/ml) and incubate overnight at 43 0 C. Identification and characterization of cointegrates 1. Use PCR to check 8 to 10 individual colonies for the presence of the modified BAC (cointegrate). Successful modification of the BAC will allow for the amplification of two specific PCR products. The first product is generated through the use of a forward 5 cointegrate primer (a sequence located upstream of the 5 end of the A-box) and a reverse 3 marker primer (EGFP, Cre). The second product is generated through the use of a forward of 5 primer of R6kг ori and a reverse 3 cointegrate primer (a sequence located downstream of the 3 end of the A-box). 2. Once successfully modified BAC clones are identified, they should be transferred to a master plate. This plate is used for latter amplification, rechecks, etc. 3. From the master plate, pick a tiny portion of a confirmed colony and inoculate 3 ml of Luria Broth medium supplemented with chloramphenicol (20 ug/ml) and ampicillin (50 ug/ml). Incubate for 8 h at 37 0 C with shaking at 300 rpm. 4. Mix 800 ul overnight culture with 200 ul 100% glycerol and store at C. 5. The modified BACs can be further confirmed by Southern blot analysis if needed. Prepare DNA by standard alkaline method and digest with unique restriction enzymes. Compare the resulting pattern with those from wild-type BAC and shuttle vector DNA by Southern blot analysis.

7 Use the A box as a hybridization probe. The correct cointegrates hybridize to two fragments due to the introduction of an additional restriction site with the recombination cassette. The wild-type BAC and the shuttle vector hybridize to only one fragment. Primers for Identification and characterization of cointegrates Reverse (3 ) EGFP: CGCCCTCGCCGGACACGCTGAAC Reverse (3 ) Cre: CAACTTGCACCATGCCGCCCACGAC Forward (5 ) R6K gamma: CAGGTTGAACTGCTGATCTTCAGATCC 2. BAC DNA Purification for Transgenesis Double acetate precipitation and CsCl gradient method Shiaoching Gong General considerations Never vortex cells or DNA suspensions. Use wide bore pipette tips to avoid damaging DNA during solution transfer. Exercise caution when handling ethidium bromide; a potent mutagen. 1. Pick a single colony of transformed bacteria from a freshly streaked chloramphenicol (20 ug/ ml) and ampicillin (50 ug/ ml) agar plate; inoculate 3 ml of Luria Broth medium containing chloramphenicol and ampicillin (same conc as agar). Incubate at 37 0 C for 8 hours. 2. Transfer 15l-50 μl of inoculated broth (depends on the cell density) into 500 ml of Luria Broth containing chloramphenicol and ampicillin (conc as above); incubate at 30 0 C for hours 3. Spin down the bacteria at 4552 g for 30 mins at 4 0 C (J6-MI Beckman- Coulter centrifuge, JS-4.2 rotor). Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained. 4. Resuspend cells in 40 ml of 10 mm EDTA, ph 8.0 by pipetting and transfer to a 250 ml bottle. 5. Add 80 ml of alkaline lysis solution (0.2N NaOH in 1% SDS: 2 ml of 10N NaOH, 10 ml of 10% SDS into 88 ml dh 2 O). Mix by *very* gently swirling and incubate for 5 min at RT. 6. Add 60 ml of cold 2M KOAc (50 ml of 7.5M KOAc, 23 ml of glacial acetic acid and 127 ml of dh 2 O, stored at 4 0 C). Mix by *very* gently

8 swirling and incubate on ice for 5 min. Spin at g for 30 mins at 4 0 C (J-25I Beckman Avanti centrifuge, JLA rotor). 7. Transfer supernatant into a 250 ml bottle, add 180 ml of isopropanol. Mix by gently swirling. Spin at 4552 g for 30 mins at 4 0 C (J6-MI centrifuge, JS-4.2 rotor). Decant the supernatant. 8. Dissolve the DNA pellet in 18 ml of 10:50 TE (1ml of 1M Tris, 10 ml of 0.5M EDTA into 89 ml dh 2 O). Add 9 ml of 7.5M KOAc and mix and incubate at C for 30 min. 9. Thaw solution and centrifuge at 4355 g for 10 mins at 4 0 C (J-25I Beckman Avanti centrifuge, JA rotor). 10. Transfer supernatant to a new tube and add 2.5 volume of ethanol. Spin at g for 30 mins at 4 0 C to precipitate the DNA (J-25i Beckman Avanti centrifuge, JLA rotor). 11. Decant supernatant and gently resuspend pellet (while still moist) in 4.4 ml of TE. Dissolve, as best possible, 10.2 g of CsCl in another 4.4 ml of TE. *Gently* mix CsCl solution with 4.4 ml of DNA until the CsCl has dissolved. Add 0.2 ml ethidium bromide solution (10 mg/ ml dh 2 0) and mix immediately. Spin at 4552 g for 10 mins at 4 0 C to remove debris (J6- MI centrifuge, JS-4.2 rotor). 12. Remove the supernatant and load into a Beckman Quick-Seal tube (16 x 76 mm, #342413) using a syringe and 18-gauge needle. Seal tubes *carefully* and place in a NVT65 rotor. (It is very important to equilibrate the tubes to be centrifuged in opposing positions: weigh them very carefully to make sure they do not differ by more than 0.05g). Spin at341,650 g overnight (>8 hours) at 18 0 C. 13. Remove tubes from rotor carefully, taking care not to disturb the gradient. Use a 23-gauge needle to poke a hole in the top of the tube. Utilizing a UV light, carefully remove the band (choose bottom band if there are two) with an 18-gauge needle with the needle bevel up. Take the band and no more (usually about 200 μl). Transfer it to a 15 ml tube and bring it up to 2 ml with TE. Extract 4-5 times with NaCl-saturated butanol (20 ml of 3M NaCl in 100 ml of butanol) until there is no more orange color. (To extract add equal volume of NaCl-saturated butanol to TE solution, mix gently, let mixture sit 30 sec to allow for separation, remove and discard top layer.) 14. Add 1 ml of H 2 O to DNA solution and then volumes of EtOH and mix. Place at C for 30 mins. Spin solution at g for 30 mins at 4 0 C to precipitate the DNA (J-25i Beckman Avanti centrifuge, JA rotor). Resuspend DNA in 0.5 ml of 0.3M NaOAc. Transfer DNA to 1.5 ml Eppendorf tube and add 1 ml EtOH. Spin down the DNA

9 at g for 30 mins at 4 0 C (Eppendorf microcentrifuge model 5417R). Discard the supernatant, fill the tube with 70% EtOH and allow the tube sit at room temperature for 5 mins. Spin the DNA again as in previous step but shorten time to 10 mins. Dry the pellet at RT for 1 min; use paper towel to get rid of the trace amount of ethanol. Resuspend DNA gently in μl of TE. Place DNA in 37 0 C incubator for min. You are likely to get 5-20 ug of BAC DNA. You should store BAC DNA at 4 0 C. (Do not store it at C!!!) 15. Check the DNA on pulse field gel and determine the concentration. 16. Digest the BAC DNA with PI-SceI (New England Biolabs, Cat # R0696S): Add together 5 10 μl (about 100 ng) of BAC DNA, 2 μl of PI-SceI enzyme, 5 μl of 10x buffer and dh 2 O to produce a final volume of 50 μl. Incubate in a 37 0 C incubator for 3 to 4 hours. 17. To dialyze the DNA, start by placing 20 ml of injection buffer (recipe below) into a sterile Petri dish and float a 25 mm, um filter (Millipore, Cat. # VSWP02500) on top with the shiny side up. Load the 50 μl of digested DNA on the top of the filter and cover the Petri dish with lid. Allow set-up to sit at RT for 4-6 hours. Transfer the DNAcontaining solution on top of the filter to a microcentrifuge tube and add enough injection buffer to return solution to original volume of 50 μl. 18. Check the DNA on pulse field gel again to confirm the concentration.

10 Injection buffer for BAC 10 mm Tris, ph mm EDTA 100 mm NaCl Primers for genotyping tail DNA The PCR primers for EFGP Lines: EGFP-5 CCTACGGCGTGCAGTGCTTCAGC EGFP-3 CGGCGAGCTGCACGCTGCCGTCCTC The PCR primers for Cre Lines: Cre-5 GGACATGTTCAGGGATCGCCAGGCG Cre-3 GCCAGATTACGTATATCCTGGCAGCG References 1. Gong, S. Yang, X.W., Li, C. and Heintz, N. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6K gamma origin of replication. Genome Res 12, (2002). 2. Gong, S., C. Zheng, M. Doughty, K. Losos, N. Didkovsky, U. B. Schambra, N. J. Nowak, A. Joyner, G. Leblanc, M.E. Hatten and N. Heintz (2003). A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425: Gong, S., M. Doughty, C. R. Harbaugh, A. Cummins, M. E. Hatten, N. Heintz and C. R. Gerfen. Targeting Cre Recombinase to Specific Neuron

11 Populations With Bacterial Artificial Chromosome Constructs. J. Neurosci, In press.

VDL101.3 CLONING TRANSGENE INTO pad5f35

VDL101.3 CLONING TRANSGENE INTO pad5f35 Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to pad5/f35. 1.2. The starting material is 10 μg plasmid DNA. 1.3. This procedure is routinely performed

More information

ITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector

ITS Sequencing in Millepora. 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector Page 1 of 5 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector 1. Digest 1 µg of pbluescript with Eco RI 2. Following digestion, add 0.1 volumes of 3M sodium acetate (ph

More information

Transformation (The method of CaCl 2 )

Transformation (The method of CaCl 2 ) PROTOCOLS E. coli Transformation (The method of CaCl 2 ) Ligation PRODUCTION competent cells of E. COLI. (Rubidium cells). Gel DNA Recovery Kit Electroporation Digestiones Plasmid purification (The method

More information

VDL100.2 CLONING TRANSGENE INTO padenox

VDL100.2 CLONING TRANSGENE INTO padenox 1. Purpose 1.1. The purpose of this protocol is to transfer a transgene from the pshuttlex plasmid to padenox. 1.2. The starting material is 10 μg plasmid DNA. 1.3. This procedure is routinely performed

More information

Alkaline Lysis Large Scale Plasmid Preparation

Alkaline Lysis Large Scale Plasmid Preparation Alkaline Lysis Large Scale Plasmid Preparation 1. Set up 10 ml overnight culture. 2. Add overnight to 500 mls of sterile LB with appropriate selective agent (e.g amp, tet...) 3. Incubate at 37 C with shaking

More information

Plasmid Maxiprep Plus Purification Kit

Plasmid Maxiprep Plus Purification Kit Plasmid Maxiprep Plus Purification Kit Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only 1 Description: The Plasmid Maxiprep Plus Purification Kit provides simple,

More information

Plasmid Maxiprep Plus Purification Kit. Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only

Plasmid Maxiprep Plus Purification Kit. Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only Plasmid Maxiprep Plus Purification Kit Cat. # : DP01MX-P10/ DP01MX-P20 Size : 10/20 Reactions Store at RT For research use only 1 Description: The Plasmid Maxiprep Plus Purification Kit provides simple,

More information

AuPreP Plasmid Midi Kit Cat. #: PMD12-143LT (25 preps/kit) PMD12-145LT (50 preps/kit)

AuPreP Plasmid Midi Kit Cat. #: PMD12-143LT (25 preps/kit) PMD12-145LT (50 preps/kit) AuPreP Plasmid Midi Kit Cat. #: PMD12-143LT (25 preps/kit) PMD12-145LT (50 preps/kit) AuPreP Plasmid Midi Kit is designed for rapid isolation of plasmid DNA of superior quality from an average of 25-100

More information

Polymerase Chain Reaction

Polymerase Chain Reaction Polymerase Chain Reaction Amplify your insert or verify its presence 3H Taq platinum PCR mix primers Ultrapure Water PCR tubes PCR machine A. Insert amplification For insert amplification, use the Taq

More information

FosmidMAX DNA Purification Kit

FosmidMAX DNA Purification Kit Cat. No. FMAX046 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 204 10/2012 1 EPILIT204 Rev. A

More information

Plasmid Midiprep Plus Purification Kit. Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only

Plasmid Midiprep Plus Purification Kit. Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only Plasmid Midiprep Plus Purification Kit Cat. # : DP01MD-P10/ DP01MD-P50 Size : 10/50 Reactions Store at RT For research use only 1 Description: The Plasmid Midiprep Plus Purification Kit provides simple

More information

Plasmid Midiprep Purification Kit

Plasmid Midiprep Purification Kit Plasmid Midiprep Purification Kit Cat. # : DP01MD/ DP01MD-100 Size : 20/100 Reactions Store at RT For research use only 1 Description: The Plasmid Midiprep Purification Kit provides simple rapid protocol

More information

Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol

Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol Generation of Gene Targeting Vectors using Recombineering - Small scale (single tube) protocol Reagent information All TSA bacterial liquid cultures are grown in: 1xTerrific Broth (TB) supplemented with

More information

BACMAX DNA Purification Kit

BACMAX DNA Purification Kit Cat. No. BMAX044 Connect with Epicentre on our blog (epicentral.blogspot.com), Facebook (facebook.com/epicentrebio), and Twitter (@EpicentreBio). www.epicentre.com Lit. # 212 10/2012 1 EPILIT212 Rev. A

More information

Presto Mini Plasmid Kit

Presto Mini Plasmid Kit Instruction Manual Ver. 03.06.17 For Research Use Only Presto Mini Plasmid Kit PDH004 (4 Preparation Sample Kit) PDH100 (100 Preparation Kit) PDH300 (300 Preparation Kit) Advantages Sample: 1-7 ml of cultured

More information

Hurricane Miniprep Kit PROTOCOL

Hurricane Miniprep Kit PROTOCOL Hurricane Miniprep Kit PROTOCOL Description: The Hurricane Miniprep Kit is designed for purification of up to 25 ug of high purity plasmid DNA from a starting volume of 2-5 ml of bacterial culture. The

More information

Recombineering Manual

Recombineering Manual Recombineering Manual Anthony Popkie The Phiel Laboratory The Research Institute at Nationwide Childrenʼs Hospital 1 BAC Transformation BACs may be transformed into either DY380, EL250 or EL350 cells.

More information

QIAfilter Plasmid Midi Kit (Cat #: 12243)

QIAfilter Plasmid Midi Kit (Cat #: 12243) QIAfilter Plasmid Midi Kit (Cat #: 12243) Things to do before starting Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase A (centrifuge briefly before use) per bottle of Buffer

More information

Geneaid Maxi Plasmid Kit & Geneaid Maxi Plasmid Kit (Endotoxin Free)

Geneaid Maxi Plasmid Kit & Geneaid Maxi Plasmid Kit (Endotoxin Free) Geneaid Maxi Plasmid Kit & Geneaid Maxi Plasmid Kit (Endotoxin Free) PM002, PME02 (2 Preparation Sample Kit) PM010, PME10 (10 Preparation Kit) PM025, PME25 (25 Preparation Kit) Instruction Manual Ver.

More information

I-Blue Midi Plasmid Kit. I-Blue Midi Plasmid Kit. (Endotoxin Free) IBI SCIENTIFIC. Instruction Manual Ver For Research Use Only.

I-Blue Midi Plasmid Kit. I-Blue Midi Plasmid Kit. (Endotoxin Free) IBI SCIENTIFIC. Instruction Manual Ver For Research Use Only. Instruction Manual Ver. 05.11.17 For Research Use Only I-Blue Midi Plasmid Kit & I-Blue Midi Plasmid Kit (Endotoxin Free) IB47180, IB47190 (2 Preparation Sample Kit) IB47181, IB47191 (25 Preparation Kit)

More information

E.Z.N.A. BAC/PAC Maxi Kit. D preps D preps

E.Z.N.A. BAC/PAC Maxi Kit. D preps D preps E.Z.N.A. BAC/PAC Maxi Kit D2154-00 2 preps D2154-01 5 preps April 2013 E.Z.N.A. BAC/PAC Maxi Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4

More information

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 8 Rev 05/0/03 EZ-0 Genomic DNA Kit Handbook Table of Contents Introduction Limitations of Use Features Applications

More information

GET Plasmid DNA 96 Well

GET Plasmid DNA 96 Well 187PR G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name GET Plasmid DNA 96 Well For High Yield & Quality Plasmid DNA Extraction (Cat. # 786

More information

Rapid amplification of cdna ends (RACE)

Rapid amplification of cdna ends (RACE) Rapid amplification of cdna ends (RACE) Rapid amplification of cdna ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE

More information

DNA miniprep by Alkaline Lysis (activity)

DNA miniprep by Alkaline Lysis (activity) DNA miniprep by Alkaline Lysis (activity) Contents 1 Alkaline Lysis 2 Exercise 1: Plasmid DNA Mini-Prep by Alkaline Lysis 3 Identification of Plasmid DNA 4 Exercise 2: Restriction Digestion Identification

More information

Purification and Characterization of a DNA Plasmid Part A CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008

Purification and Characterization of a DNA Plasmid Part A CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008 Purification and Characterization of a DNA Plasmid Part A CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008 INTRODUCTION DNA Plasmids. A plasmid is a small double-stranded, circular DNA molecule

More information

Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning

Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content

More information

E.Z.N.A. Endo-free Plasmid DNA Mini Kit I

E.Z.N.A. Endo-free Plasmid DNA Mini Kit I E.Z.N.A. Endo-free Plasmid DNA Mini Kit I D6948-00 5 preps D6948-01 50 preps D6948-02 200 preps E.Z.N.A. Endo-free Plasmid DNA Mini Kit II D6950-00 5 preps D6950-01 50 preps D6950-02 200 preps May 2013

More information

GENERAL BIOLOGY LABORATORY II

GENERAL BIOLOGY LABORATORY II Weeks 9-10: Bioassays of major biomolecules: Nucleic acids GENERAL BIOLOGY LABORATORY II Canbolat Gürses, Hongling Yuan, Samet Kocabay, Hikmet Geckil Department of Molecular Biology and Genetics Inonu

More information

Storage and Stability

Storage and Stability Contents Introduction....................................................... 2 Kit Contents...................................................... 3 Before Starting....................................................

More information

E.Z.N.A. Endo-free Plasmid DNA Mini Kit I

E.Z.N.A. Endo-free Plasmid DNA Mini Kit I E.Z.N.A. Endo-free Plasmid DNA Mini Kit I D6948-00 5 preps D6948-01 50 preps D6948-02 200 preps E.Z.N.A. Endo-free Plasmid DNA Mini Kit II D6950-00 5 preps D6950-01 50 preps D6950-02 200 preps November

More information

Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions

Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions Plasmid DNA Isolation Column Kit Instruction Manual Catalog No. SA-40012: 50 reactions SA-40011: 100 reactions Maxim Biotech, Inc. 780 Dubuque Avenue, So. San Francisco, CA 94080, U.S.A. Tel: (800) 989-6296

More information

Plasmid Transformation

Plasmid Transformation Plasmid Transformation Zhang Junqi, Han Wendong 2012-10-11 1 1. Experiments: Transformation of Plasmid DNA 2. Demonstration: Plasmid extraction 2 Characteristics of plasmids Plasmid is a type of DNA existence

More information

pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20

pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20 pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20 1 Kit Contents Contents pgm-t Cloning Kit pgm-t Vector (50 ng/μl) 20 μl T4 DNA Ligase (3 U/μl) 20 μl 10X T4 DNA Ligation Buffer 30 μl

More information

Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis *

Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis * Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Jeroen D. Langereis * Laboratory of Pediatric Infectious Diseases, Department of Pediatrics and Laboratory of Medical Immunology,

More information

Prepare NIB containing 2mg/ml of Zymolyase (Zymolyase may not completely dissolved. Use as suspension)

Prepare NIB containing 2mg/ml of Zymolyase (Zymolyase may not completely dissolved. Use as suspension) S. pombe 2D gel analysis E. Noguchi Reagents for DNA preparation NIB (Nuclear Isolation Buffer) : 500mL 17% glycerol 60% 142mL 50mM MOPS buffer (m.w.:209.3) 5.23g 150mM potassium acetate 1M 75mL 2mM magnesium

More information

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK

EZ-10 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK EZ-0 SPIN COLUMN GENOMIC DNA MINIPREPS KIT HANDBOOK (Bacteria, Plant, Animal, Blood) Version 5.0 Rev 03/25/205 Table of Contents Introduction 2 Limitations of Use 2 Features 2 Applications 2 Storage 2

More information

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix

Cat # Box1 Box2. DH5a Competent E. coli cells CCK-20 (20 rxns) 40 µl 40 µl 50 µl x 20 tubes. Choo-Choo Cloning TM Enzyme Mix Molecular Cloning Laboratories User Manual Version 3.3 Product name: Choo-Choo Cloning Kits Cat #: CCK-10, CCK-20, CCK-096, CCK-384 Description: Choo-Choo Cloning is a highly efficient directional PCR

More information

E.Z.N.A. HP Plasmid DNA Mini Kit I

E.Z.N.A. HP Plasmid DNA Mini Kit I E.Z.N.A. HP Plasmid DNA Mini Kit I D7043-00 5 preps V - Spin D7043-01 50 preps V - Spin E.Z.N.A. HP Plasmid DNA Mini Kit II D7045-00 5 preps V - Spin D7045-02 200 preps V - Spin July 2013 E.Z.N.A. HP

More information

7.13 Experimental Microbial Genetics

7.13 Experimental Microbial Genetics MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms. 7.13 Fall 2008 Page

More information

E. cloni EXPRESS Electrocompetent Cells

E. cloni EXPRESS Electrocompetent Cells E. cloni EXPRESS Electrocompetent Cells FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX:

More information

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA)

Figure 1. Map of cloning vector pgem T-Easy (bacterial plasmid DNA) Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 6: Ligation & Bacterial Transformation (Bring your text and laptop to class if you wish to work on your assignment during

More information

UltraClean Midi Plasmid Prep Kit

UltraClean Midi Plasmid Prep Kit UltraClean Midi Plasmid Prep Kit Catalog No. Quantity 12700-20 20 Preps Instruction Manual Please recycle Version: 05232014 1 Table of Contents Introduction... 3 Protocol Overview... 3 Flow Chart... 4

More information

EasyPrep TM Plasmid Maxiprep Manual

EasyPrep TM Plasmid Maxiprep Manual EasyPrep TM Plasmid Maxiprep Manual Catalog#: PD05-01, PD05-02 PD05-11, PD05-12 Bioland Scientific LLC 14925 Paramount Blvd., Suite C Paramount, CA 90723 USA Tel: (877) 603-8882 Fax: (562) 733-6008 Email:

More information

PCR Cloning Protocol. Day 1: Isolation of MG1655 genomic DNA (adapted from Current Protocols in Mol. Biol. (1997), Supplement 27.

PCR Cloning Protocol. Day 1: Isolation of MG1655 genomic DNA (adapted from Current Protocols in Mol. Biol. (1997), Supplement 27. Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning STUDENTS SHOULD USE A P-2 MICROPIPETTOR FOR ALL VOLUMES 2 µl. Day 1: Isolation of MG1655 genomic DNA (adapted from Current Protocols in

More information

Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab

Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab Gateway Cloning Protocol (Clough Lab Edition) This document is a modification of the Gateway cloning protocol developed by Manju in Chris Taylor's lab With the Gateway cloning system, a PCR fragment is

More information

E.Z.N.A. Plasmid DNA Mini Kit I

E.Z.N.A. Plasmid DNA Mini Kit I E.Z.N.A. Plasmid DNA Mini Kit I D6942-00 5 preps Q - Spin D6942-01 50 preps Q - Spin D6942-02 200 preps Q - Spin D6943-00 5 preps V - Spin D6943-01 50 preps V - Spin D6943-02 200 preps V - Spin E.Z.N.A.

More information

(Overexpression) Product Manual. Cat#: SB-P-AV reactions. Store at -80 C upon arrival

(Overexpression) Product Manual. Cat#: SB-P-AV reactions. Store at -80 C upon arrival (Overexpression) Product Manual FOR RESEARCH USE ONLY Not for clinical and therapeutic use Cat#: SB-P-AV-103-01 10 reactions Store at -80 C upon arrival AdenoONE TM - Cloning and Expression Kit FROM TRANSGENE

More information

Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli

Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli Electrocomp GeneHogs E. coli One Shot Electrocomp GeneHogs E. coli Catalog nos. C8080-10, C8080-03, C800-05 Version E 17 May 2007 25-0387 www.invitrogen.com Overview Introduction The information in this

More information

Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive

Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive Designing and creating your gene knockout Background The rada gene was identified as a gene, that when mutated, caused cells to become hypersensitive to ionizing radiation. However, why these mutants are

More information

TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0

TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 Cat. # 9760 For Research Use TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 Product Manual Table of Contents I. Description... 3 II. Kit Components... 3 III. Shipping and Storage... 4 IV. Preparation

More information

Application of Molecular Biology tools for cloning of a foreign gene

Application of Molecular Biology tools for cloning of a foreign gene IFM/Kemi Linköpings Universitet September 2013/LGM Labmanual Project course Application of Molecular Biology tools for cloning of a foreign gene Table of contents Introduction... 3 Amplification of a gene

More information

TIANpure Mini Plasmid Kit

TIANpure Mini Plasmid Kit TIANpure Mini Plasmid Kit For purification of molecular biology grade DNA www.tiangen.com PP080822 TIANpure Mini Plasmid Kit Kit Contents Storage Contents RNaseA (10 mg/ml) Buffer BL Buffer P1 Buffer P2

More information

Purification of DNA Oligonucleotides

Purification of DNA Oligonucleotides Purification of DNA Oligonucleotides Polyacrylamide gel: In a beaker, add 30 ml 30% acrylamide solution (To make this solution, add 29 g acrylamide to 1 g bis-acrylamide, bring to 100 ml with dh 2 O) 31.5

More information

E.Z.N.A. Plasmid DNA Mini Kit I

E.Z.N.A. Plasmid DNA Mini Kit I E.Z.N.A. Plasmid DNA Mini Kit I D6942-00 5 preps Q - Spin D6942-01 50 preps Q - Spin D6942-02 200 preps Q - Spin D6943-00 5 preps V - Spin D6943-01 50 preps V - Spin D6943-02 200 preps V - Spin E.Z.N.A.

More information

E.Z.N.A. Soil DNA Kit. D preps D preps D preps

E.Z.N.A. Soil DNA Kit. D preps D preps D preps E.Z.N.A. Soil DNA Kit D5625-00 5 preps D5625-01 50 preps D5625-02 200 preps December 2016 E.Z.N.A. Soil DNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing

More information

Bacterial Transformation: Unlocking the Mysteries of Genetic Material

Bacterial Transformation: Unlocking the Mysteries of Genetic Material PR009 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Bacterial Transformation: Unlocking the Mysteries of Genetic Material Teacher s Guidebook

More information

PCR Cloning Protocol

PCR Cloning Protocol Modifications to EXPERIMENTS 21 and 24: PCR and Molecular Cloning This experiment was designed by Dylan Dodd, based on research completed in Dr. Isaac Cann s lab*, with modifications and editing of content

More information

PROTOCOL 1: EXTRACTION OF DNA FROM BACTERIA

PROTOCOL 1: EXTRACTION OF DNA FROM BACTERIA PROTOCOL 1: EXTRACTION OF DNA FROM BACTERIA The basic standard procedures for isolation of bacterial DNA are based on lysozyme digestion of the cell wall, detergent lysis, disruption of protein-nucleic

More information

Samples are frozen cell pellets stored in the -80 C freezer and contain ~5 x 10 8 cells from growth experiments.

Samples are frozen cell pellets stored in the -80 C freezer and contain ~5 x 10 8 cells from growth experiments. Boiling RNA Preparation: for RNA isolation (from cell pellets). Modified by Jiyeon Kim on June 6, 2011. From Sarah Young, s protocol and RNeasy Mini Handbook. Media to have: Lysis Buffer (1.2 ml), make

More information

Adult Neuron Isolation Protocol (R. Kaletsky 9/2014)

Adult Neuron Isolation Protocol (R. Kaletsky 9/2014) Notes before starting: Adult Neuron Isolation Protocol (R. Kaletsky 9/2014) Begin protocol ~45min before sort time to minimize time between cell harvesting and sorting Prepare pronase solution immediately

More information

UltraClean 6 Minute Mini Plasmid Prep Kit

UltraClean 6 Minute Mini Plasmid Prep Kit UltraClean 6 Minute Mini Plasmid Prep Kit Catalog No. Quantity 12300-50 50 Preps 12300-100 100 Preps 12300-250 250 Preps Instruction Manual Introduction Use the UltraClean 6 Minute Mini Plasmid Prep Kit

More information

ProductInformation. Genomic DNA Isolation Kit. Product No. GDI-3 Technical Bulletin No. MB-275 May 2000 TECHNICAL BULLETIN

ProductInformation. Genomic DNA Isolation Kit. Product No. GDI-3 Technical Bulletin No. MB-275 May 2000 TECHNICAL BULLETIN Genomic DNA Isolation Kit Product No. GDI-3 Technical Bulletin No. MB-275 May 2000 TECHNICAL BULLETIN ProductInformation Product Description Sigma s Genomic DNA Isolation Kit isolates genomic DNA from

More information

Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH

Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH Capsule deletion via a λ-red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578 Tzu-Wen Huang 1,2, Irene Lam 2, Hwan-You Chang 3, Shih-Feng Tsai 1, Bernhard

More information

INDEX INDEX 0 KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 IMPORTANT NOTES 2 EUROGOLD TOTAL RNA ISOLATION PROTOCOL 2 DNA CONTAMINATION 5

INDEX INDEX 0 KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 IMPORTANT NOTES 2 EUROGOLD TOTAL RNA ISOLATION PROTOCOL 2 DNA CONTAMINATION 5 INDEX INDEX 0 KIT COMPONENTS 1 STORAGE AND STABILITY 1 INTRODUCTION 1 IMPORTANT NOTES 2 EUROGOLD TOTAL RNA ISOLATION PROTOCOL 2 DNA CONTAMINATION 5 QUANTITATION AND STORAGE OF RNA 5 RNA QUALITY 5 TROUBLESHOOTING

More information

Bio-Rad Laboratories, Inc Alfred Nobel Dr. Hercules, CA USA (510) Rev C

Bio-Rad Laboratories, Inc Alfred Nobel Dr. Hercules, CA USA (510) Rev C Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA (510) 741-1000 1-800-424-6723 4110111 Rev C Aurum Plasmid Mini Kit Instruction Manual For technical service, call your local Bio-Rad

More information

BRED: Bacteriophage Recombineering with Electroporated DNA

BRED: Bacteriophage Recombineering with Electroporated DNA BRED: Bacteriophage Recombineering with Electroporated DNA Introduction We have developed a system for generating mutations in lytically replicating mycobacteriophages that we have termed BRED: Bacteriophage

More information

Molecular Techniques Third-year Biology

Molecular Techniques Third-year Biology PLANNING Genetics Lab practices Molecular Techniques. Genetics Lab practices protocol. 2015-16 PCR-DIRECTED MUTAGENESIS, MOLECULAR CLONING AND RESTRICTION ANALYSIS Sessions 1 & 2 (2x3 hours): PCR-directed

More information

Introduction. Principle

Introduction. Principle Contents Introduction............................................................ 2 Principle.............................................................. 2 Storage and Stability.....................................................

More information

EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST

EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST EXPERIMENT 4 CLONING THE UPSTREAM REGION OF THE GENE OF INTEREST Purpose: To determine the activity of the promoter of the gene of interest at the cellular and tissue levels in Arabidopsis plants via the

More information

E.Z.N.A. Soil DNA Kit. D preps D preps D preps

E.Z.N.A. Soil DNA Kit. D preps D preps D preps E.Z.N.A. Soil DNA Kit D5625-00 5 preps D5625-01 50 preps D5625-02 200 preps April 2013 E.Z.N.A. Soil DNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing

More information

Bacterial Transformation and Protein Purification

Bacterial Transformation and Protein Purification Bacterial Transformation and Protein Purification Group 4 Natalie Beale Gregory A. Pate Justin Rousseau Dohee Won Introduction The purpose of this experiment is to perform a genetic transformation and

More information

E.Z.N.A. Tissue RNA Kit. R preps R preps

E.Z.N.A. Tissue RNA Kit. R preps R preps E.Z.N.A. Tissue RNA Kit R6688-00 5 preps R6688-01 50 preps May 2015 E.Z.N.A. Tissue RNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Important Notes...4 Homogenization

More information

Subcloning 1. Overview 2. Method

Subcloning 1. Overview 2. Method Subcloning 1. Overview: 1.1. Digest plasmids for vector + insert 1.2. CIP treat vector 1.3. Gel purify vector + insert 1.4. Ligate 1.5. Transform bacteria with ligation reactions 1.6. Qiagen or Eppendorf

More information

EZ-10 SPIN COLUMN HANDBOOK

EZ-10 SPIN COLUMN HANDBOOK EZ-0 SPIN COLUMN HANDBOOK EZ-0 Spin Column Plasmid DNA Mini Kit EZ-0 Spin Column PCR Products Purification Kit EZ-0 Spin Column DNA Gel Extraction Kit Version 20.0 Rev 3/23/205 ISO900 Certified 20 Konrad

More information

Fast and reliable purification of up to 100 µg of transfection-grade plasmid DNA using a spin-column.

Fast and reliable purification of up to 100 µg of transfection-grade plasmid DNA using a spin-column. INSTRUCTION MANUAL ZymoPURE Plasmid Miniprep Kit Catalog Nos. D4209, D4210, D4211 & D4212 (Patent Pending) Highlights Fast and reliable purification of up to 100 µg of transfection-grade plasmid DNA using

More information

Easy Tissue & Cell Genomic DNA Purification Kit. Cat. #:DP021E/ DP021E-150 Size:50/150 reactions Store at RT For research use only

Easy Tissue & Cell Genomic DNA Purification Kit. Cat. #:DP021E/ DP021E-150 Size:50/150 reactions Store at RT For research use only Easy Tissue & Cell Genomic DNA Purification Kit Cat. #:DP021E/ DP021E-150 Size:50/150 reactions Store at RT For research use only 1 Description: The Easy Tissue & Cell Genomic DNA Purification Kit is ideal

More information

DNA Extraction DNA Extraction (small scale) using CTAB method

DNA Extraction DNA Extraction (small scale) using CTAB method DNA Extraction DNA Extraction (small scale) using CTAB method This method is relatively simple, and has been used successfully with a wide range of monocot and dicot species. The method may be used with

More information

Mag-Bind Ultra-Pure Plasmid DNA 96 Kit. M x 96 preps M x 96 preps

Mag-Bind Ultra-Pure Plasmid DNA 96 Kit. M x 96 preps M x 96 preps M1258-00 1 x 96 preps M1258-01 4 x 96 preps December 2015 Table of Contents Introduction...2 Kit Contents/Storage and Stability...3 Preparing Reagents...4 Plasmid Protocol with Lysate Clearance via Centrifugation...5

More information

E.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps

E.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps E.Z.N.A. Yeast Plasmid Mini Kit D3376-00 5 preps D3376-01 50 preps November 2015 E.Z.N.A. Yeast Plasmid Mini Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing

More information

Creating pentr vectors by BP reaction

Creating pentr vectors by BP reaction Creating pentr vectors by BP reaction Tanya Lepikhova and Rafael Martinez 15072011 Overview: 1. Design primers to add the attb sites to gene of interest 2. Perform PCR with a high fidelity DNA polymerase

More information

Data Sheet Quick PCR Cloning Kit

Data Sheet Quick PCR Cloning Kit Data Sheet Quick PCR Cloning Kit 6044 Cornerstone Ct. West, Ste. E DESCRIPTION: The Quick PCR Cloning Kit is a simple and highly efficient method to insert any gene or DNA fragment into a vector, without

More information

E.Z.N.A. FastFilter Plasmid DNA Midi Kit

E.Z.N.A. FastFilter Plasmid DNA Midi Kit E.Z.N.A. FastFilter Plasmid DNA Midi Kit D6905-00 2 preps D6905-03 25 preps D6905-04 100 preps E.Z.N.A. FastFilter Plasmid DNA Maxi Kit D6924-00 2 preps D6924-01 5 preps D6924-03 25 preps D6924-04 100

More information

LIBRARY SCREENING. For background reading, read Maniatis or Current Protocols.

LIBRARY SCREENING. For background reading, read Maniatis or Current Protocols. LIBRARY SCREENING Overview. Screening a l library involves the following steps: titering the library to determine the PFU (plaque forming units per ml of library stock), primary plating of the library,

More information

Specifications. Kit Specifications. Alcohol Precipitation: Up to 100 ml Column Purification: Up to 5 ml Column Binding Capacity 25 µg

Specifications. Kit Specifications. Alcohol Precipitation: Up to 100 ml Column Purification: Up to 5 ml Column Binding Capacity 25 µg 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com BAC DNA MiniPrep Kit Product # 18050 Product Insert The BAC DNA

More information

Part I: Plasmid Construction

Part I: Plasmid Construction Part I: Plasmid Construction Q5 polymerase was used for PCR amplification of assembly fragments, while Pfu polymerase was used to perform PCR site-directed mutagenesis. Gel extraction or PCR purification

More information

Bacteria Genomic DNA Purification Kit

Bacteria Genomic DNA Purification Kit Bacteria Genomic DNA Purification Kit Cat #:DP025/ DP025-150 Size:50/150 reactions Store at RT For research use only 1 Description: The Bacteria Genomic DNA Purification Kit provides a rapid, simple, and

More information

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual

Cold Fusion Cloning Kit. Cat. #s MC100A-1, MC101A-1. User Manual Fusion Cloning technology Cold Fusion Cloning Kit Store the master mixture and positive controls at -20 C Store the competent cells at -80 C. (ver. 120909) A limited-use label license covers this product.

More information

Hetero-Stagger PCR Cloning Kit

Hetero-Stagger PCR Cloning Kit Product Name: Code No: Size: DynaExpress Hetero-Stagger PCR Cloning Kit DS150 20 reactions Kit Components: Box 1 (-20 ) phst-1 Vector, linearized Annealing Buffer Ligase Mixture phst Forward Sequence Primer

More information

Aurum Plasmid Mini Kit. Instruction Manual. Bio-Rad Laboratories, Inc Alfred Nobel Dr. Hercules, CA USA (510)

Aurum Plasmid Mini Kit. Instruction Manual. Bio-Rad Laboratories, Inc Alfred Nobel Dr. Hercules, CA USA (510) Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA (510) 741-1000 1-800-424-6723 Aurum Plasmid Mini Kit Instruction Manual For technical service, call your local Bio-Rad office, or

More information

BRED: Bacteriophage Recombineering with Electroporated DNA

BRED: Bacteriophage Recombineering with Electroporated DNA Phagehunting Program BRED: Bacteriophage Recombineering with Electroporated DNA Introduction We have developed a system for generating mutations in lytically replicating mycobacteriophages that we have

More information

Kits for isolation of plasmid DNA in medium scale

Kits for isolation of plasmid DNA in medium scale PLASMID MIDI KIT PLASMID MIDI ENDOTOXIN-FREE Cat. No. EM16 / EM17 Version: 1.2014 Kits for isolation of plasmid DNA in medium scale EXTRACTME is a registered trademark of BLIRT S.A. PLASMID MIDI KIT PLASMID

More information

pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases For research use only Cat. # : GVT202 Size : 20 Reactions

pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases For research use only Cat. # : GVT202 Size : 20 Reactions pgm-t Cloning Kit For direct cloning of PCR products generated by Taq DNA polymerases Cat. # : GVT202 Size : 20 Reactions Store at -20 For research use only 1 pgm-t Cloning Kit Cat. No.: GVT202 Kit Contents

More information

Amgen Protocol: Introduction and a few comments:

Amgen Protocol: Introduction and a few comments: Amgen Protocol: Introduction and a few comments: The following is a shortened version of the Amgen Lab. This series of labs involves the creation of a recombinant plasmid, subsequent transformation of

More information

Linköpings Universitet. Site-directed mutagenesis of proteins

Linköpings Universitet. Site-directed mutagenesis of proteins IFM/Kemi August2011/LGM Linköpings Universitet Site-directed mutagenesis of proteins Competent E. coli cells Site-specific mutagenesis Analysis on agarose gel Transformation of plasmids in E. coli Preparation

More information

InterLab Study: Plasmid amplification

InterLab Study: Plasmid amplification igem TU/e 2015 Biomedical Engineering Eindhoven University of Technology Room: Ceres 0.04 Den Dolech 2, 5612 AZ Eindhoven The Netherlands Tel. no. +31 50 247 55 59 2015.igem.org/Team:TU_Eindhoven InterLab

More information

Nematostella vectensis genomic DNA preparation and Telomeric Restriction Fragment Analysis (TRF)

Nematostella vectensis genomic DNA preparation and Telomeric Restriction Fragment Analysis (TRF) Daniel Lackner Host lab: Martindale lab / Kewalo Marine Lab Dates of visit: 2/4/2012 2/18/2012 Nematostella vectensis genomic DNA preparation and Telomeric Restriction Fragment Analysis (TRF) Rationale

More information

Low-cost DNA extraction for use in TILLING and Ecotilling assays

Low-cost DNA extraction for use in TILLING and Ecotilling assays Low-cost DNA extraction for use in TILLING and Ecotilling assays Version 1.3 (January 31, 2013) Plant Breeding and Genetics Laboratory Prepared by Bernhard Hofinger and Bradley Till 1. MATERIALS Company

More information

Low cost and non-toxic genomic DNA extraction for use in molecular marker studies.

Low cost and non-toxic genomic DNA extraction for use in molecular marker studies. Low cost and non-toxic genomic DNA extraction for use in molecular marker studies. Version 1.4, February 28 th, 2013. Prepared by Bernhard Hofinger, Owen Huynh and Brad Till. 1. OBJECTIVE To develop and

More information

E.Z.N.A. Plasmid DNA Midi Kit

E.Z.N.A. Plasmid DNA Midi Kit E.Z.N.A. Plasmid DNA Midi Kit D6904-00 2 preps D6904-03 25 preps D6904-04 100 preps E.Z.N.A. Plasmid DNA Maxi Kit D6922-00 2 preps D6922-01 5 preps D6922-02 20 preps D6922-04 100 preps July 2015 E.Z.N.A.

More information