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Bad fragment amplification


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4 replies to this topic

#1 criscastells

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Posted 21 November 2013 - 10:39 AM

Hi everybody,

 

I have design a pair of primers for ACTN1 that amplify a fragment of 188bp. During a year, in all the PCR I have performed I obtained a 188bp fragment but the last time I use them the fragment was about 150bp. Thinking about a problem with the primers I prepare new aliquots but I still obtain a band of 150bp. Any idea about what can happen? I thought about different transcripts but in primer Blast I only found amplification for 188bp.

 

Thanks you very much for your help in advance.

 

CRIS



#2 phage434

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Posted 21 November 2013 - 10:42 AM

Two possibilities:

1) Your DNA marker has changed, not your PCR product

2) The salt concentration of your PCR product has changed, leading to different running speeds.

 

Were you purifying your DNA prior to gel running, either before or after the change?

Have you switched ladders? Or switched buffers for your ladders?



#3 criscastells

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Posted 21 November 2013 - 10:48 AM

I do not purify my DNA product but I have change the ladder. Anyway the other fragments I have been studied (GJA1, RPL22 between others) do not change.



#4 hobglobin

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Posted 21 November 2013 - 11:48 AM

Do you still have former samples left that you amplified with the size 188 bp? If you would run them again you could exclude (or proof) the possibility that running conditions cause the problem.


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#5 phage434

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Posted 21 November 2013 - 12:40 PM

Perhaps your template has changed.






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