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Team:Northwestern/Protocol/Quikchange (from primers to colonies!
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Timsterxox (Talk | contribs) (New page: ==Primers== I use [http://mekentosj.com/enzymex/ enzyme x] to work with DNA sequences, it is installed on all the macs and has nice translation and reverse complement functions. Many of ...) |
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==Primers== | ==Primers== | ||
| - | + | We use [http://mekentosj.com/enzymex/ enzyme x] to work with DNA sequences, it is installed on all the macs and has nice translation and reverse complement functions. Many of the same functions are available in [http://www.biology.utah.edu/jorgensen/wayned/ape/ a plasmid editor], which has Mac, Windows, and Linux versions. | |
*Forward Primer | *Forward Primer | ||
| Line 16: | Line 16: | ||
*When they arrive make a 20uM stock | *When they arrive make a 20uM stock | ||
| - | I use netprimer to make sure the primer Tm is good. You should have a rating above 70 (above 80 is preferable) for your primers. | + | I use netprimer to make sure the primer Tm is good. You should have a rating above 70 (above 80 is preferable) for your primers. |
| - | + | ||
==PCR== | ==PCR== | ||
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*order PAGE purified primers | *order PAGE purified primers | ||
| + | Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]] | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
Latest revision as of 01:27, 23 October 2010
Contents |
Primers
We use [http://mekentosj.com/enzymex/ enzyme x] to work with DNA sequences, it is installed on all the macs and has nice translation and reverse complement functions. Many of the same functions are available in [http://www.biology.utah.edu/jorgensen/wayned/ape/ a plasmid editor], which has Mac, Windows, and Linux versions.
- Forward Primer
- identify the bases that code for your residue of interest (eg. 295-297)
- A - Copy the 10 bases before the codon (eg. 285-294)
- B - Write your new codon after the 10 bases you just copied
- C - Copy the 22 bases that follow the codon (eg. 298-319), I always terminate in a G or C
- your forward primer should be 5'-A-B-C-3'
- Reverse Primer
- D - Copy the ten bases after the codon (eg. 298-307) - then reverse complement it!
- E - Write your new codon - then reverse complement it!
- F - Copy the 22 bases that precede the codon (eg. 273-294) - then reverse complement it!
- your reverse primer should be 5'-D-E-F-3'
- When they arrive make a 20uM stock
I use netprimer to make sure the primer Tm is good. You should have a rating above 70 (above 80 is preferable) for your primers.
PCR
Recipe
- 1.25ul of 20uM Primer F
- 1.25ul of 20uM Primer R
- 10ul Phusion Buffer (5x)
- 1ul of 10mM dNTPs
- 0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine)
- 1.0 ul Phusion polymerase
- H2O to 50uL
Cycle
- 98C for 30s
- 98C for 5s
- 53C for 20s
- 72C for 20s/kb (usually plasmids are ~7kb = 2:20)
- Cycle 16 times - more cycles are actually bad!
- 72C for 8:00
- 4C for hold
If you are using a different polymerase the annealing/extension/denaturing times and temperatures will be different!!
DpnI and transformation
- After PCR, add 1ul of DpnI to each pcr tube
- Incubate 1 hour-O/N at 37C
- PCR Purify using Qaigen kit
- Add 5ul of PCR reaction into 25ul DH5alpha cells
- Incubate on ice for 5 minutes
- Heatshock at 42C for 45 seconds
- Recover on ice for 2 minutes
- add 200ul LB (if in 96 well or 8 strip format) to each reaction
- shake at 37C for 1 hour
- plate on warmed antibiotic plates
If it doesn't work
- Miniprep more colonies... ;)
- order PAGE purified primers
Back to Protocol
