PCR trouble - (Oct/03/2005 )

Hi everybody,

I am trying to amplify a 1970 bp fragment form chicken cDNA.My primer's Tms are 59,5 and 62.Firstly I used 59 C as annealing temp.but I saw bands at 500, 700, 750 bp on agarose instead of 1970.Then I tried touchdown PCR from 62 to 60 and it worked.I saw my 1970 bp fragment( but not bright, very weak) but not alone, with other 500, 700, 750 bp fragments again.So I extracted 1970 bp band from gel and did another PCR from this fragment but I saw 500, 700, 750 bp band again!!

I think my primers anneal somewhere inside 1970 bp so they amplify smaller regions like 500, 700, 750??

Do you have any comments?

Thank you...

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It´s very likely.... If you´re trying to clone the fragment, then you may try to amplify several times, gel purify, restriction digest and clone. That could be all you need. But if you need great quantities...ummm, you may need new primers!

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Thanks for your advice:)

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Sometimes the problem is the Taq you are using not the primer - the Taq might be "falling off" before it reaches the 1970 bases...which would explain the small size products in your second PCR as well. Try using a long PCR system such as Roche's Expand High Fidelity System - i have used that one for amplyfying 2000bp quite successfully. Invitrogen also has such a system...

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hmm at the beginning I was using Roche Taq and then I started to use Fermentas Taq but the results are same.Maybe I should try an enzyme suitable for long templates.

Thanks for the answer:)

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Dear Katanin,

Since you amplified 500, 700 750bp constantly, I think your primer is not specific enough.
There are several ways to solve it.

Other wise you can try ti optimised you PCR reaction towards high stringency, such as:
1) Increase annealing temperature by using gradient annealing temperature ranging from 62-67oC
2) decrease Mg concentration to minimum level (1.5mM)
3) decrease KCl concentration by using 0.8x of 0.7x PCR buffer.

If every thing still fail you, no other choice but redesign your primer.

Could you please give me your PCR profile?


Best regards

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Hi Hadrian,

Sure I can give:) :

Amount of PCR Reagents I use is as follows:(for 25 ul total reaction volume)
10x PCR buffer (500mM KCl) ----> 2,5 ul (1x final)(without MgCl2)
MgCl2(25mM)--->1.5 ul (final 1.5 mM)
dNTP (10mM each)----> 0,5 ul
forward primer ---> 0,5 ul
reverse primer ---> 0,5 ul
enzyme (5 u/ul-Taq from fermentas)---> 0,5 ul

my PCR conditions is as follows:
94- 2min. initial denat.
94-30sec.
62-30 sec. ] 10x cycle
68- 3min.

94-30
61-30 ] 4x cycle
68-3.5

94-30
61-30 ] 4x cycle
68-4

94-30
61-30 ] 4x cycle
68-4.5

94-30
61-30 ] 4x cycle
68-5

94-30
60-30 ] 5x cycle
68-5.5

94-30
60-30 ] 9x cycle
68-6

68- 10 min.
4- final hold.

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Hi again Hadrian:)

I forgot to ask: you say I can try a gradient annealing from 62 up to 67.should I increase the temp by one by like 62-63-64-65-66-67 or can I try 62-64-67 for example? My concern is if I use all the temp. between 62-67, number of cycles per temperature will be too little and insufficient for amplification at the related temperature.Can I explain?

Thank you so much...

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Can I ask why you're running such a complicated protocol? I just successfully amplified and cloned three genes -- a 3,168 bp gene from Arabidopsis thaliana cDNA, a 3,219 bp gene from Oryza sativa cDNA, and a 3,318 bp from Human cDNA without issues, just using a straight-forward PCR and Hi-Fi Taq:

95ºC, 2 mins
95ºC, 30 secs \
55ºC, 30 secs | x 35
68ºC, 4 minutes /
68ºC, 5 minutes
4ºC, hold

I usually don't work with cDNA, (or with plant or human genes, for that matter) -- I work with bacteria. Perhaps this worked because nobody told me it wouldn't?

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Hi,

I use such a complicated protocol because I tried the others and did not work:)

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