DNA/RNA Protocols CLONING. Standard PCR set-up, per reaction. 50ul total volume/tube

Transcription

1 DNA/RNA Protocols Kahn Lab, UCLA CLONING Standard PCR set-up, per reaction 50ul total volume/tube 45ul KOD Hot Start master mix 1ul 10uM 5 primer 1ul 10uM 3 primer 1ul DMSO 1ul template DNA o 1ul ddh2o [or up to 50ul total] 1. Make master mixes of primers and/or templates when appropriate 2. After adding all components, vortex and spin down briefly before starting reaction Standard PCR Program [always use heated lid!] C, 2 minutes C, 15 seconds C, 15 seconds [annealing temperature depends on primer Tm] C, 1 minute [extension time depends on amplicon size] 5. go to step 2, 29 more times 6. 4 C, t=0 [forever] 7. end Phenol Chloroform Extraction For extracting and purifying DNA after PCR 1. Transfer PCR reaction to a 0.6ml microcentrifuge tube [50ul] 2. Add 50ul ddh2o to the same tube 3. Add 100ul phenol-chloroform-isoamyl alcohol mix [volume is now 200ul] 4. Vortex, spin down at 10000rpm for 5 minutes in a 1.5ml tube without lid 5. After spin, you will see 2 phases; take 90ul of the top phase and transfer to a clean 1.5ml microcentrifuge tube 6. Add 10ul 3M sodium acetate, ph 5.0 [or 3M potassium acetate, ph 5.6] 7. Add 250ul 200-proof ethanol 8. Vortex, spin down at rpm for 20 minutes 9. Using a 10ul tip on a Pasteur pipet, aspirate all liquid without disturbing the pellet 10. Add 250ul ice-cold 70% ethanol [made from pure ethanol]

2 11. No need to vortex; spin down at 10000rpm for 10 minutes 12. Carefully aspirate all liquid without disturbing the pellet by holding the Pasteur pipet/tip at the 200ul mark on the 1.5ml tube 13. You should now have a dry DNA pellet! Restriction Endonuclease Digestion, per sample For digesting PCR products after phenol chloroform extraction, and plasmid DNA, before gel extraction and ligation 40ul total volume/tube [preferably in PCR tube] 4ul NEB 10x buffer 0.4ul BSA Master Mix 2ul enzyme 1 39ul total 2ul enzyme 2 [if necessary] 30.6ul ddh2o [or 32.6ul if using 1 enzyme] 1ul plasmid DNA* [or add 1ul ddh2o to DNA pellet] *refers to maxiprepped DNA. Ideally, try to digest 1 2ug of DNA; if using miniprepped DNA, adjust the DNA and ddh2o volumes accordingly. 1. Vortex master mix well and spin down briefly before adding to DNA 2. Vortex reactions well and spin down briefly before placing at 37 C 3. Digest for 1 4 hours at 37 C, depending on the enzymes; standard digestion is 1 hour 4. Can digest samples using the thermocycler; programs 37-1HR, 37-2HR, 37-3HR, and 37-4HR all end with 4 C forever Antarctic Phosphatase Treatment For removing phosphate groups from the ends of a vector directly after enzyme digestion, and preventing vector-only re-ligation; necessary when ligating products cut with only 1 enzyme 1. Add 4.5ul 10x Antarctic Phosphatase buffer to each 40ul-enzyme digest 2. Add 1ul Antarctic Phosphatase enzyme to each digest [total volume is now 45.5ul] 3. Run ANTPHOS5 PCR program: 37 C, 15 minutes; 65 C, 10 minutes; 4 C forever 4. Once the 65 C enzyme inactivation step is done, proceed to gel extraction

3 Making Agarose Gels Agarose gels are measured in percent weight by volume; this means that a 1% gel contains 1g of agarose in 100ml TAE buffer Use a 2% gel for resolving small DNA fragments <1kb [genotyping, small PCR products] Use a 0.7% gel for resolving larger DNA fragments [usually 1 12kb but best for 5+ kb] Use a 1% gel for resolving DNA fragments between 1 and 5kb if 0.7% doesn t provide great resolution 1. Measure an appropriate amount of agarose for the DNA fragment size you want to analyze 2. Add an appropriate volume of 1X TAE buffer to agarose in a 500ml Erlenmeyer flask a. For small gels, use 60ml; for large gels, use 110ml b. Small gels i. 0.7% = 0.42g agarose + 60ml TAE ii. 2% = 1.2g agarose + 60ml TAE c. Large gels i. 0.7% = 0.77g agarose + 110ml TAE ii. 2% = 2.2g agarose + 110ml TAE 2. Swirl flask to mix, and heat in microwave for 45sec 2min [long enough for agarose to dissolve]; be careful not to let it boil over! 3. Allow to cool slightly, then add ul ethidium bromide to agarose solution; swirl to mix 4. Pour gel into casting apparatus [gel box, tray, multi-well comb] and allow to set 5. When gel has hardened, carefully remove comb, lift gel tray out of the box and turn sideways to expose gel edges to the buffer chambers 6. Add 1X TAE to gel box; make sure the top and bottom buffer chambers are full, and that the top of the gel is covered with TAE as well 7. Add 1ul ethidium bromide to the bottom buffer chamber & mix by pipetting 8. After loading ladders and samples, put the lid on, plug the cables into the power supply, and run at ~200V a. Always make sure that the black electrode is at the top and the red electrode is at the bottom of the gel b. Negatively-charged DNA molecules will migrate towards the positive charge [red electrode] Run to RED 9. Once the loading dye has migrated far enough, unplug the cables, remove the gel tray and view it on a UV transilluminator or gel imager

4 Gel Extraction/Purification For separating DNA fragments after enzyme digestion and before ligation 1. Pour an agarose/etbr gel using the thicker side of a wide-well comb [use an appropriate agarose concentration for the fragment size you want to purify] 2. Run out the samples on the gel as usual 3. After taking a picture of the gel using the longer wavelength [less damaging to the DNA], cut out the bands you want to purify using clean razorblades and clean 1.5ml tubes 4. Proceed to gel purification using the instructions provided in the kit, and elute in 30ul Ligation & Transformation For ligating desired vectors and inserts, and transforming into competent E.coli cells Prewarm LB-agar + antibiotic plates at 37 C before ligation; use one plate for each ligation [don t forget vector-only control plates!] 11ul total volume/tube 2ul 10x ligase buffer [use small aliquots; make sure it smells!] Master Mix 3ul ddh2o 6ul total 1ul T4 DNA ligase 1ul vector 4ul insert [or ddh2o for vector-only control] 1. Vortex master mix well and spin down briefly before adding to DNA 2. Vortex reactions well and spin down briefly 3. Ligate for 5 minutes at RT 4. In the meantime, thaw Z-competent cells on ice 5. After cells are thawed and ligation is complete, add 1ul of each ligation reaction to a 50ul-aliquot of Z-competent cells [on ice] 6. DO NOT VORTEX! Instead, gently finger-tap to mix 7. Place on ice for 5 10 minutes 8. Plate entire volume of cells and ligated DNA onto LB-agar + antibiotic plates; use glass beads to spread cells 9. Incubate plates upside down at 37 C overnight

5 Picking Colonies & Miniprepping 1. Inoculate each colony with a pipet tip into 2ml LB broth + 100ug/ml ampicillin, in a 14ml BD tube 2. Usually you will need to pick multiple colonies per plate 3. With the cap loosely on, grow cultures at 37 C overnight, shaking at 250rpm 4. The following day, use 1.5ml of overnight culture for miniprepping; save 500ul in the fridge to start a maxiprep culture later 5. Pellet the culture and proceed with plasmid purification according to the miniprep kit protocol; elute in 50ul Diagnostic Restriction Endonuclease Digestion, per sample For digesting plasmid DNA, to confirm construct identity [after ligations, mini- and maxipreps] 10ul total volume/tube [in PCR tube] 1ul NEB 10x buffer 0.1ul BSA Master Mix 0.25ul enzyme 1 9ul total 0.25ul enzyme 2 [if necessary] 7.4ul ddh2o [or 7.65ul if using 1 enzyme] 1ul plasmid DNA* *refers to miniprepped DNA. Ideally, try to digest 250ng of DNA; if using maxiprepped DNA, adjust the DNA and ddh2o volumes accordingly. 1. Vortex master mix well and spin down briefly before adding to DNA 2. Vortex reactions well and spin down briefly before placing at 37 C 3. Digest for 1 4 hours at 37 C, depending on the enzymes; standard digestion is 1 hour [try to use the same digestion time that you used before ligation] 4. Can digest samples using the thermocycler; programs 37-1HR, 37-2HR, 37-3HR, and 37-4HR all end with 4 C forever 5. After digestion, run out on an agarose/etbr gel to check products

6 Sequencing: Premixed Samples for Laragen For confirming construct identity after miniprep digests 6ul total volume/pcr strip tube 1ul 10uM primer [10pmol/ul; 5 OR 3 ; do not combine primers!] ng DNA [often ~3-5ul miniprepped DNA] up to 6ul ddh2o 1. Make a master mix of primers and/or DNA whenever possible 2. Assign each DNA/primer combo a simple name or number; label PCR tubes on the SIDE [not the lid!] 3. Enter Individual DNA Sequencing Requests online at a. Username: kahnlab; Password: daniel b. Service: Premix c. Payment Type: Enter a PO number [previously obtained from the OBGYN office] d. Reaction Mix Order: 0, Chemistry: BigDye-V3.1 e. Comments: 5dollars per sample for premixed as per Jin f. Use the same Template names as the ones on the tubes g. DNA type: Plasmid; Primer: Custom, assign a simple name [preferably part of the same sample name]; 10pmol/ul h. Purification Method: Other [for Bioland] 4. After submitting the request, print out 2 copies of the confirmation; one for your notebook and one to go along with the bag of samples 5. Laragen will pick up the samples from our lab between 2:00-3:00pm the same day [if submitted before 1:30pm] or the next day [if submitted after 1:30pm] 6. Results will be available within a day of pickup and can be downloaded online

7 Genotyping from Mouse Tail/Ear Biopsies 1. Add 50ul tail base buffer [25mM NaOH, 0.2 mm EDTA] to each tail/ear piece, in PCR tubes a. Use 100ul buffer for DTR biopsies 2. Place samples at 95 C for 45 minutes, then 4 C forever 95C program on thermocycler a. For DTR genotyping: use 100C-20M program instead 3. Afterwards, add 50ul neutralization buffer [40 mm TrisHCl ph 5.5] to each sample & mix well by pipetting 4. Use neutralized samples as the template for PCR Genotyping PCR set-up, per reaction 25ul total volume/tube 12.5ul Accustart PCR Supermix [Quanta] + 10ul ddh2o [preferred] OR 22.5ul Platinum PCR Supermix [Invitrogen] 1ul primer mix o for gender: use mix of 4 10uM primer stocks: SRY and Fabpi, 5 and 3 o for OVA: use mix of 4 10uM primer stocks: OVA and Fabpi, 5 and 3 o for DTR: use mix of 10uM 5 and 3 DsRed primers 2ul template DNA [neutralized tail DNA] 1. Make a master mix of primers and supermix when appropriate 2. Add 23ul primer/supermix mixture to 2ul template DNA 3. After adding all components, vortex and spin down briefly before starting reaction Genotyping PCR Programs: always use heated lid! PCR-TM55 for gender genotyping C, 2 minutes C, 15 seconds C, 15 seconds [annealing temperature depends on primer Tm] C, 1 minute [extension time depends on amplicon size] 5. go to step 2, 29 more times 6. 4 C, t=0 [forever] 7. end TM60-2 for OVA genotyping

8 DTR-GENO for DTR/DsRed genotyping C, 2 minutes C, 30 seconds C, 30 seconds [annealing temperature depends on primer Tm] C, 1 minute [extension time depends on amplicon size] 5. Go to step 2, 39 more times C, 7 minutes 7. 4 C, t=0 [forever] 8. end Pour a 2% agarose/etbr gel using the thicker side of a small-well comb After PCR, run the entire sample volume out on the gel All samples with Fabpi primers should have a Fabpi band between 400 and 500bp Males genotyped for gender will also have an SRY band just below 300bp OVA mice will also have an OVA band ~300bp DsRed/DTR mice will have a band ~500bp cdna Synthesis from RNA Standard set-up, per sample 20ul total volume/sample 4ul 5x qscript reaction mix Master Mix 1ul qscript RT enzyme 5ul total 1000ng RNA RNA volume up to 15ul H2O 15ul total 1. Make master mixes when appropriate 2. Use filter tips and RNAse-free tubes for RNA work 3. After adding all components, vortex and spin down briefly before starting reaction 4. Run RT program on thermocycler: a. 22 C, 5 minutes b. 42 C, 30 minutes c. 85 C, 5 minutes d. 4 C, t=0 [forever] 5. After reaction is done, add 180ul of water to sample to bring volume up to 200ul; use this diluted cdna as template for qpcr 6. Store leftover cdna at -20 C

9 qrt-pcr Standard qpcr set-up, per well 25ul total volume/well 12.5ul 2x Perfecta SYBR master mix Master Mix 0.5ul 10uM primer mix [5 and 3 ] 20ul total 7ul H2O [or 10ul H2O if using 2ul cdna] 5ul cdna [or 2ul cdna if you don t have a lot of sample] After diluting cdna to 200ul total with H2O and making master mixes [2x mix, primer mix, H2O]: 1. Add DNA and master mix to each well [can use multichannel when convenient] 2. Seal plate thoroughly 3. Spin down plate ~25 seconds, vortex, spin down again 4. Run qpcr program a. Usually, use qpcr Tm 59 templ with melt program in the Kahn folder: i. 95 C, 2 minutes ii. 95, 15 seconds iii. 59 C, 45 seconds iv. repeat ii and iii for 40 cycles v. 95 C, 15 seconds vi. 59 C, 15 seconds vii. Melt curve gradient: 20 minutes viii. 95 C, 15 seconds ix. 4 C, hold 5. Or store at 4 C until ready to run, and spin briefly before run