Mutagenesis: Difference between revisions
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updated for new PCR program and to use Pfu Turbo as the polymerase |
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*Template (dilute to ~ 0.1 mg/mL | *Template (dilute to ~ 0.1 mg/mL | ||
*Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program | *Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program | ||
*dNTP's (2 mM; add 10 uL of each dNTP (100 mM from invitrogen) into 460 uL water, and make 50 uL aliquots) | |||
*PFU Turbo | *PFU Turbo | ||
*DpnI | *DpnI | ||
| Line 13: | Line 14: | ||
##5 uL of dNTP mix | ##5 uL of dNTP mix | ||
##28 uL of water | ##28 uL of water | ||
#Add 1 uL of Pfu | #Add 1 uL of Pfu Turbo | ||
#Run PCR Program ( | #Run PCR Program (Mutagenesis 8/15/20 minute, depending on template) | ||
##95C for 30s | ##95C for 30s | ||
##Repeat 18 cycles: | ##Repeat 18 cycles: | ||
##95C for 30s | ##95C for 30s | ||
##55C for 1 min | ##55C for 1 min | ||
##68C for 1 min/kb plasmid length (8 min default) | ##68C for 1 min/kb plasmid length (8 min default, but use 15 or 20 min for larger templates.) | ||
#Place on ice for 2 min | #Place on ice for 2 min | ||
#Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C. | #Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C. | ||
| Line 29: | Line 30: | ||
#Spread out entire transformation on appropriate antibiotic | #Spread out entire transformation on appropriate antibiotic | ||
#Grow O/N at 37C | #Grow O/N at 37C | ||
#Pick a colony, miniprep and sequence to verify mutation | #Pick a colony, miniprep and sequence to verify mutation | ||
#[[Check Clones for Correct Mutation]] | |||
Latest revision as of 15:15, 13 January 2010
Materials
- Template (dilute to ~ 0.1 mg/mL
- Primers (prepare by adding 2 uL of each sense and antisense primer + 196 uL water and label as 1 uM primer mix). Design using Stratagene Primer Design Program
- dNTP's (2 mM; add 10 uL of each dNTP (100 mM from invitrogen) into 460 uL water, and make 50 uL aliquots)
- PFU Turbo
- DpnI
- Supercompetent XL1-Blue Cells (Stratagene)
Protocol
- Mix:
- 5 uL 10X Reaction Buffer
- ~20 ng Template (1 uL of minprep)
- 10 uL of Primer Mix
- 5 uL of dNTP mix
- 28 uL of water
- Add 1 uL of Pfu Turbo
- Run PCR Program (Mutagenesis 8/15/20 minute, depending on template)
- 95C for 30s
- Repeat 18 cycles:
- 95C for 30s
- 55C for 1 min
- 68C for 1 min/kb plasmid length (8 min default, but use 15 or 20 min for larger templates.)
- Place on ice for 2 min
- Add 1 uL DpnI, mix and spin down. Digest 1h or overnight at 37C.
- Thaw XL1-Blue cells on ice and make 50 uL aliquots (round tube)
- Add 1 uL of digest to cells and mix.
- Incubate 30 min on ice.
- Heat at 42 C for 45 s, then place on ice for 2 min.
- Add 450 uL SOC preheated at 42C and grow 1h at 37C shaking
- Spread out entire transformation on appropriate antibiotic
- Grow O/N at 37C
- Pick a colony, miniprep and sequence to verify mutation
- Check Clones for Correct Mutation