PCR: cDNA works on 18S, but not Gap or designed primers - (May/31/2010 )

Hi there. I'm trying to work with some primers i designed.

I tried doing a regular pcr with the cDNA and 3 sets of designed primers.
None of these worked.

I also tried my cDNA on regular GapDh primers for a control.
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I also tried using a plasmid with my GapDh primers and doing a pcr with that, and i got two bands appearing on the gel with the plasmid, whereas i got none with the cDNA.

My guess was that the cDNA was just not viable, but i tested it with a nanodrop system, and it showed it to be quite viable (around 3000ng/ul in the stock. I had diluted this to make 100ug samples which were used in the PCR. But checking these also showed themselves to be fine.)

At the suggestion of my supervisor, i tried using the rt-pcr with my cdna and 18s primers. This seemed to give appropriate bands when ran out on a gel. (But i may be jumping the gun here if i cant get a regular pcr to work.)

What should i try now if my PCR with a gap control isnt working, but my cDNA seems to be fine?

Hi there,
we do a lot of real-time PCR in our lab so I hope I can help:

first question: what is your source for your RNA/cDNA??
second: I hope you are not using 100ug but rather 100ng for your reaction! otherwise you titered out your primers and other resources in the reaction.

third: what enzyme are you using? some need activation for 2min at 95C others don't! depends on if they are inhibited chemically or with an antibody
forth: what is the melting temperature for your primers?? rule of thumb: G/C 4C, A/T 2C mismatch by percentage..

forth: your cycle times seem incredibly long...we use the ABI machine for real-time but you seem to only want to do regular PCR...95C 15 sec is usually fine..don't fry your enzyme...60C for 30 sec...for annealing sounds good..but depends on your annealing temp for primers..you might want to try 55C!-15sec might be enough , 68C for 20sec sounds fine-yet depends on how long the template is you want to PCR... depends on the fitness of the enzyme you use...usually 1min/1kb..if proof-reading probably longer...check manufacturing recommendations...
the 68C for 5 min is at the end of all cycles to deactivate the enzyme-right? not every step...unless you want to clone it is optional!

there is a lot of potential to optimize..Mg concentration is first...or run a heat gradient on a regular PCR machine (55-65C for example over 10 samples),
can also adjust primer as well as nucleotide conc but usually not necessary (magnesium conc. first 3-5 mM)

for more advice would need more details but hope it gives you a starting point for further trouble shooting


cheers

blackmouse on Jun 9 2010, 09:08 AM said:

Hi there,
we do a lot of real-time PCR in our lab so I hope I can help:

first question: what is your source for your RNA/cDNA??
second: I hope you are not using 100ug but rather 100ng for your reaction! otherwise you titered out your primers and other resources in the reaction.

third: what enzyme are you using? some need activation for 2min at 95C others don't! depends on if they are inhibited chemically or with an antibody
forth: what is the melting temperature for your primers?? rule of thumb: G/C 4C, A/T 2C mismatch by percentage..

forth: your cycle times seem incredibly long...we use the ABI machine for real-time but you seem to only want to do regular PCR...95C 15 sec is usually fine..don't fry your enzyme...60C for 30 sec...for annealing sounds good..but depends on your annealing temp for primers..you might want to try 55C!-15sec might be enough , 68C for 20sec sounds fine-yet depends on how long the template is you want to PCR... depends on the fitness of the enzyme you use...usually 1min/1kb..if proof-reading probably longer...check manufacturing recommendations...
the 68C for 5 min is at the end of all cycles to deactivate the enzyme-right? not every step...unless you want to clone it is optional!

there is a lot of potential to optimize..Mg concentration is first...or run a heat gradient on a regular PCR machine (55-65C for example over 10 samples),
can also adjust primer as well as nucleotide conc but usually not necessary (magnesium conc. first 3-5 mM)

for more advice would need more details but hope it gives you a starting point for further trouble shooting


cheers