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When I amplify 50ng of DNA template using PCR, I don't see any primer dimer or unamplified template band on my agarose gel. When I amplify 10ng of DNA template, I see primer dimer and unamplified template bands. What do these results show?
There could be sooo many reasons why this isn't working .. so I suggest that you run through a checklist.
1. Re-Check your primers sequences with your template (like, start all over again a re-derive your primers.. and double check them! .. I've been PCR'in for years and I screwed up my primers last month and wasted an entire week !!!)
2. Check the concentrations of everything!
- Having excess DNA template, primers tend to be
- Excess DNTP's haven't been problematic for me .. but try
sticking to the manufacturers recommendation for each
- CHECK your MgCl2 (MgSO4) concentrations!!!! (this is
Critical too little will prevent amplification, too much
causes non-specific amplification ... higher taq error
3. Throw away your Water!!! Get a fresh batch of dH2O in a recently autoclaved container. OR (Better-yet) use the dH2O from another molecular kit ... (the manufactures triple-sterilize their water) You'd be suprised to know how often water screws-up PCR.
5. Make sure your taq is kept on ICE whenever you take it out of the fridge ... it will not work correctly if left at room-temp.
6. Check your primer annealing temperature!!! (if it's too high the primers will not anneal... if it's too low you'll get non-specific amplification and maybe dimerization).. let me know if you need help with estimating the temperature.
7. When you set up your reaction make sure you have add everything!!
- 1. DNA template
- 2. dH2O
- 3. 10X buffer (w/o MgCL2 or MgSO4)
- 4. MgCL2 (or MgSO4)
- 5. Forward primer (5')
- 6. Reverse primer (3')
- 7. DNTP's
- 8. Taq (or Hi-Fi Taq) polymerase
8. If none of this works .. you should start replace all reagents!!! (new Taq, new DNTP's, New 10x buff., new MgCL2 ... primers and Template tend to be stable so those probably won't need to be replaced)
2 posts • Page 1 of 1
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