Lidstrom:Colony PCR: Difference between revisions
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
==One Recipe:== | |||
* for 25 uL: 12.5 uL 2X master mix, 0.5 uL 10 uM forward primer, 0.5 uL 10 uM reverse primer, 11.5 uL H2O | |||
* for 20 uL: 10 uL 2X master mix, 0.4 uL 10 uM forward primer, 0.4 uL 10 uM reverse primer, 9.2 uL H2O | |||
cycling: | |||
# 5 min at 95oC (once) | |||
# 1 min at 95oC | |||
# 1.5 min at 55 oC (annealing) | |||
# 1 min/kb at 68oC (OneTaq extension temp: use the one for your polymerase) | |||
# repeat steps 2-5 30-40 times | |||
# 5 min at 72oC (once) | |||
# 4oC forever | |||
==General:== | ==General:== | ||
*Use Taq. | *Use Taq if you aren't going to use the DNA you amplify for a construct. Taq is cheap. Do not use Phusion (~$1/50uL rxn). Taq is less accurate & less fast but that's fine. | ||
*The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC. | *The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC. | ||
*The primer concentration varies in different people's recipes. [[User:Janet B. Matsen|Janet]] hasn't tested variations. | |||
*The primer concentration varies in different people's recipes. [[Janet B. Matsen|Janet]] hasn't tested variations. | |||
*One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used. | *One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used. | ||
*If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion. | *If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion. | ||
*[[Janet B. Matsen|Janet]] is using NEB's [http://www.neb.com/nebecomm/products/productM0486.asp OneTaq] quick-load (not hot start) & their recommended [http://www.neb.com/nebecomm/appNotes/appnoteM0486.pdf | *[[User:Janet B. Matsen|Janet]] is using NEB's [http://www.neb.com/nebecomm/products/productM0486.asp OneTaq] quick-load (not hot start) & their recommended [http://www.neb.com/nebecomm/appNotes/appnoteM0486.pdf protocol] | ||
** manual: Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm | |||
==Parameters:== | ==Parameters:== | ||
*T_anneal: | *T_anneal: up to you. For VF2 & VR 54-56oC works well. | ||
*t_anneal: use 30s/kb and round up. | *t_anneal: use 30s/kb and round up. | ||
*rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing. | *rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing. | ||
==Comments== | |||
*It might be tempting to make a 1X soltuion with primers included, store it in the freezer, and thaw it as you need to use it. The problem with this: primer dimers may amplify. Obviously the degree to which this is an issue will depend on your primers, but it is likely worth avoiding by sticking with the 2X freezer stock. Also, there may be issues with the protein's stability upon freezing in a lower buffer/stabilizer concentration. | |||
**From Justin Siegel 2/6/2012: "The problem isn't the associate primers, it that when they associate and the polymerase extends them. Then your reaction can get overrun since now extended primer dimers can act a perfect template in future amplifications and will preferably amplify over larger pieces. So if there is primers+enzyme but no template you run a really high risk of getting primer dimers." | |||
**For what it is worth, my mix that includes the primers VF2 & VR works great. -[[User:Janet B. Matsen|Janet]] 2/7/2012 | |||
*Stay tuned for an image of what "overloading" can look like! | *Stay tuned for an image of what "overloading" can look like! | ||
Revision as of 14:31, 17 April 2013
Back to Protocols
One Recipe:
- for 25 uL: 12.5 uL 2X master mix, 0.5 uL 10 uM forward primer, 0.5 uL 10 uM reverse primer, 11.5 uL H2O
- for 20 uL: 10 uL 2X master mix, 0.4 uL 10 uM forward primer, 0.4 uL 10 uM reverse primer, 9.2 uL H2O
cycling:
- 5 min at 95oC (once)
- 1 min at 95oC
- 1.5 min at 55 oC (annealing)
- 1 min/kb at 68oC (OneTaq extension temp: use the one for your polymerase)
- repeat steps 2-5 30-40 times
- 5 min at 72oC (once)
- 4oC forever
General:
- Use Taq if you aren't going to use the DNA you amplify for a construct. Taq is cheap. Do not use Phusion (~$1/50uL rxn). Taq is less accurate & less fast but that's fine.
- The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC.
- The primer concentration varies in different people's recipes. Janet hasn't tested variations.
- One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used.
- If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion.
- Janet is using NEB's OneTaq quick-load (not hot start) & their recommended protocol
- manual: Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm
Parameters:
- T_anneal: up to you. For VF2 & VR 54-56oC works well.
- t_anneal: use 30s/kb and round up.
- rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing.
Comments
- It might be tempting to make a 1X soltuion with primers included, store it in the freezer, and thaw it as you need to use it. The problem with this: primer dimers may amplify. Obviously the degree to which this is an issue will depend on your primers, but it is likely worth avoiding by sticking with the 2X freezer stock. Also, there may be issues with the protein's stability upon freezing in a lower buffer/stabilizer concentration.
- From Justin Siegel 2/6/2012: "The problem isn't the associate primers, it that when they associate and the polymerase extends them. Then your reaction can get overrun since now extended primer dimers can act a perfect template in future amplifications and will preferably amplify over larger pieces. So if there is primers+enzyme but no template you run a really high risk of getting primer dimers."
- For what it is worth, my mix that includes the primers VF2 & VR works great. -Janet 2/7/2012
- Stay tuned for an image of what "overloading" can look like!
