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PCR Mutagenesis R334C
PCR Mutagenesis R334C
Tube (labeled R334C 10ng/uL):
9.0uL MBG Water (Fridge A)
1.0uL 10 ng/uL DNA
Diluted from wtCFTR_pGHE 100ng/uL (Freezer C, Box 6)
1.25uL 10uM R334C_CFTR_mut_FOR1
1.25uL 10uM R334C_CFTR_mut_REV1
12.5uL Q5 Mastermix (Freezer C, Box 12 Yellow tube)
Tube (labeled R334C 25ng/uL):
9.0uL MBG Water (Fridge A)
1.0uL 25 ng/uL DNA
Diluted from wtCFTR_pGHE 100ng/uL (Freezer C, Box 6)
1.25uL 10uM R334C_CFTR_mut_FOR1
1.25uL 10uM R334C_CFTR_mut_REV1
12.5uL Q5 Mastermix (Freezer C, Box 12 Yellow tube)
Spin before thermocycling!
Mutagenesis Primers:
Resuspending Primers
Locate your primers in the package and ensure they are the primers you ordered.
Compare the nucleotide sequences and GC% on each primer label to the order invoice. The melting temperature will likely differ between the label and the order (this is not important).
Spin down the tubes using the centrifuge machine. SPIN ALL TUBES BEFORE OPENING! This is because dry pellet can often come dislodged during shipping and could be in the cap.
Create a 100uM master stock by adding MBG water into the tube. Take the amount of nanomoles (nmols) on the label and multiply it by 10. That number is how many microliters of MBG water you'll add to the tube.
Added 255 uL of MBG water to 25.5nmol FOR1 primer.
Added 294 uL of MBG water to 29.4 nmol REV1 primer.
Label the top of each primer tube with its name and concentration (100 uM).
Wait 10 minutes to allow the master stock to rest at room temperature and then mix well (centrifuge) before creating the working solutions.
Store in CFTR True-Open box in Freezer C.
Thermocycling Conditions: https://docs.google.com/spreadsheets/d/18Yk2jufUmbJi62y05_AmscWo1AZ1X6ZGCj0Ejvv85P4/edit#gid=0
When thermocycling is finished (4.5 hours), transport to Fridge A until digesting with DpnI.
Dpn1 Digestion:
Pipet 2 uL solution into a new tube and into Freezer A for gel electrophoresis
wtCFTR_pGHE R334C 10ng/uL (PCR)
wtCFTR_pGHE R334C 25ng/uL (PCR)
Pipet 1 uL FD Dpn1 (Freezer A) into both tubes containing the remaining DNA solution
wtCFTR_pGHE R334C 10ng/uL (Dpn1)
wtCFTR_pGHE R334C 25ng/uL (Dpn1)
Incubate at 37°C for 16.5 hours
Ran gel at 140V for 52 minutes (EJT3122)
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