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

Transcription

1 pgm-t Cloning Kit Cat. # : GVT202 Size : 20 Reactions Store at -20 1

2 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 2X T4 Rapid Ligation Buffer 100 μl Control Fragment (300 bp) DH5α Competent Cell (100 μl/tube) Control Plasmid 5 μl 21 tubes 5 μl Colony primer pairs 400 μl 15 ml SOC medium 1 bottle Nuclease-free Water 300 μl Storage Store competent cells -70 C, store other reagents (including vector and ligation reagents) at -20 C. Aliquot reagents to prevent contamination due to repeated freezing and thawing. DH5 Genotype SupE44 lacu169 (φ80lacz M15) hsdr17 reca1 enda1 gyra96 thi-1 rela1 2

3 Introduction The pgm-t Vectors are linearized vectors with a single 3 -terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the ligation efficiency of PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases. The pgm-t Vectors are high-copy-number vectors containing T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of the enzyme β-galactosidase. Insertional inactivation of the α-peptide allows identification of recombinants by blue/white screening on indicator plates. Important Notes 1. It's necessary to use the control fragment during the transformation process for subsequent trouble shooting if encountering problems. 2. It's suggested to keep a part of the ligation products so that ligation can be carried out again if any unexpected problems occurred. 3. The volume of bacteria samples used for plating on antibiotic containing LB agar plate can be adjusted according to the experimental needs. For example, use <100 μl of sample if transformation efficiency is high, and use 200~300 μl if transformation efficiency is low. To concentrate the bacterial concentration, centrifuge at 4,000 rpm for 2 min and remove some supernatant prior to plating. Store the remaining sample at 4 C for futher use. Protocol All procedures must be done in asepsis environment. 1. If the product contains 3 -A overhangs, proceed directly to step 3. Analyze the PCR product on agarose gel before using to confirm it is the product of interest. The PCR product to be ligated can be gel-purified or purified directly from the PCR amplification using PCR cleanup & gel extraction system. Clean-up of reactions prior to ligation is recommended to remove primer dimers or other undesired reaction products to improve ligation efficiency. 3

4 2. Prepare an A-tailing reaction according to the following condition: Component purified PCR fragment 10X Reaction Buffer with MgCl 2 Volume 8 μl 1 μl 2.5 mm datp or dntp (2.5 mm each) 0.8 μl 5 u/μl Taq DNA Polymerase 0.2 μl Reaction condition: at 72 C for 30 min. The product after A-tailing procedure can be used in ligation reaction. 3. The vector must be thawed on ice. Do not freeze and thaw repeatedly. Centrifuge the tube before use. 4. Add the following components into the asepsis tube: The molar ratio of vector and insert fragment must be controlled in 1:3 ~ 1:8. Ligation reaction may be disturbed beyond this range. Component Contents in Ligation system Using 10X Ligation Buffer Reaction system Control system Using 2X Ligation Buffer Reaction system Control system PCR fragment (added A) X μl - X μl - Control fragment (300 bp, 50 ng/μl) pgm-t Vector (50 ng/μl) 10X T4 DNA Ligation Buffer 2X T4 DNA Rapid Ligation Buffer - 1 μl - 1 μl 1 μl 1 μl 1 μl 1 μl 1 μl 1 μl μl 5 μl T4 DNA Ligase 1 μl 1 μl 1 μl 1 μl Nuclease-free Water Up to 10 μl Up to 10 μl Up to 10 μl Up to 10 μl 4

5 Note: The kit is supplied with two different ligation buffers: 10X T4 DNA Ligation Buffer and 2X T4 DNA Rapid Ligation Buffer. Do not add two buffers in one tube. 5. Gently mix the solution, and centrifuge shortly. Incubate the mix at 22~26 C for an hour or at 16 C overnight if using 10X T4 DNA Ligation Buffer; incubate the mix at C for min if using 2X T4 DNA Rapid Ligation Buffer. Place the mix on ice after incubation. Note: It s recommended to incubate the mix at 16 C overnight if using 10X T4 DNA Ligation Buffer. 6. Transformation a. Prepare agar plates for transformation. Apply 80 µl IPTG (50 mm) and 80 µl X-gal (20 mg/ml) onto the surface of agar plate containing ampicillin (50 µg/ml). Spread completely using a sterile bent glass rod or a specialized spreader, and incubate the plate at 37 C shortly. b. Transformation step i. Remove tube(s) of DH5 competent cells from storage and place in an ii. iii. ice bath until just thawed. Carefully add part of ligation-reaction mixture to 50~100 µl DH5 competent cells. The volume of ligation-reaction mixture added should be one tenth the volume of competent cell. Gently flick the tubes to mix and incubate on ice for 30 min. Suggestion: Transform control plasmid to competent cells to detect transformation efficiency. Add 1 μl control plasmid to another tube with proper competent cell, and follow transformation step. Heat-shock the cells for 45 sec in a water bath at exactly 42 C (do not shake). Immediately return the tubes to ice and incubate for 2~3 min (do not shake). Add 250 μl SOC medium (brought to room temperature) per tube ( without antibiotics), and incubate for 1 hour at 37 C with shaking (~225 rpm). iv. Gently mix the medium. Plate 150 μl transformate onto each LB agar plate containing antibiotic to ensure good separation of colonies for subsequent single-colony isolation. Plate with asepsis elbow glass rod, and incubate the plate at 37 C for 12~16 hours. 5

6 7. Detection a. Quick detection: Detect whether the fragment has inserted correctly using colony PCR. i. Calculate the number of clones that are to be screened, prepare the master mix for each 25 μl PCR solution, e.g. for 10 colonies prepare 275 μl master mix (25 μl x 11). For each 25 μl PCR mixture, prepare: Component Volume Water 10X Reaction Buffer with MgCl μl 2.5 μl 2.5 mm dntp 2 μl colony primer pairs (5 μm) 2 μl 5 u/μl Taq DNA Polymerase 0.2 μl Or use PCR Master Mix Kit (Cat# RP02) Component Volume Water 5X PCR master mix colony primer pairs (5 μm) 18 μl 5 μl 2 μl ii. iii. iv. Dispense 25 μl aliquots of the master mix into the appropriate number of PCR tubes. Use a sterile 10 or 200 μl pipet tip (or a toothpick) to gently touch each colony, wash the pipette tip in 25 μl master mix. Note: Do not pick too much colony or add agar to the master mix, which may inhibit the PCR reaction. Perform the following PCR program: 6

7 Cycles Temperature Time 1 94 C 2 min C 1 min 50 C 1 min 72 C 1 min (1 min/kb) 1 72 C 5 min v. Load 2~5 μl PCR products on 1.5~2% agarose gel for electrophoresis analysis. For positive clones, the molecular weight should be 178 bp plus the inserted DNA molecular weight, e.g. for 300 bp PCR positive control, the PCR product should be about 478 bp. For self-ligation, the PCR product will be around 178 bp. b. General detection: Select a colony into 1~5 ml liquid LB culture medium (containing 50~100 μg/ml ampicillin), and culture at 37 C overnight with shaking. Use either commercially available kit (GeneMark Cat# DP01 ) or in house protocol to purify plasmid DNA. The inserted DNA can be cut off by EcoR I digestion or by other multiple cloning enzymes. See below restriction map of multiple cloning sites for appropriated enzyme selection. c. Sequencing: sequence the fragment after general or quick detection. 7

8 pgm-t Vector Map pgm-t Vector Cloning Site 8

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