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Problems with a PCR-No bands and I am starting to question my sanity

pcr sequencing

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4 replies to this topic

#1 Ladymarge

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Posted 02 February 2012 - 11:40 AM

Hello everyone,

I am having problems with this PCR, it has worked for someone else in the lab before, so this person just gave me the protocol, concentrations, I did it just like they did and it didn't work. I need to amplify the multiple cloning site of a plasmid in order to be able to sequence my insert.

I changed all the reagents, reconstituted the primers, dNTPs, MgCl2, water, buffer, got positive controls from someone else (purified plasmids)I got a new taq no so long ago. So, I did a PCR totally unrelated to this one and it worked. Yesterday, I repeated this PCR, using betaine and doing a gradient PCR and it still didn't work. I am using the following concentrations per one reaction:

*MilliQ water -25.5 ul
*Betine- 20 ul
* PCR buffer -5ul
* MgCl2 3mM- 3ul
* Primer 1 and 2 0.4 mM- 2ul each
*Polymerase (1.250 U)- 0.5ul
*DNA- 2ul (the concentration should be around 30-70nM)

Final volume: 50 ul


Thanks!!!!

#2 Ladymarge

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Posted 02 February 2012 - 11:42 AM

The melting temperature of the primers is 63C and my labmate told me that an annealing temperature of 56 worked for her. So when I did the gradient, it ranged from 46-66.

Thanks!

#3 phage434

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Posted 02 February 2012 - 01:27 PM

That seems like way too much betaine. I would cut back to 5 ul and replace remainder with water. You could try different magnesium concentrations. Are you sure your buffer doesn't already have enough magnesium?

#4 leelee

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Posted 02 February 2012 - 09:55 PM

Is your template ok?

#5 almost a doctor

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Posted 03 February 2012 - 02:17 AM

A couple of observations:

1. MilliQ water -25.5 ul + Betine- 20 ul + PCR buffer -5ul + MgCl2 3mM- 3ul + Primer 1 and 2 0.4 mM- 2ul each + Polymerase (1.250 U)- 0.5ul + DNA- 2ul (the concentration should be around 30-70nM) = 25.5 + 20 + 5 + 3 + 2 + 2 + 0.5 + 2 = 60µl and not the 50µl you state. So your final concentrations are not what you think they are. For example your PCR buffer is at 0.833x rather than 1x (assuming the stock is at 10x).

2. your reaction mix doesn't have dNTPs. Is this a typo or are they included in the PCR buffer (I've never seen this). If a typo, what's the volume and stock concentration. Again, due to the final volume being 60 and not 50 the dNTPs will be in a different concentration that the one you think.

My 2 cents





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