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PCR troubleshooting for molecular cloning

PCR troubleshooting for molecular cloning - PCR - Polymerase Chain Reaction Forum

PCR troubleshooting for molecular cloning - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 07-20-2009, 07:12 PM
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Default PCR troubleshooting for molecular cloning



Hi Everyone!

I am having problems amplifying my insert; basically, following the completion of the PCR reaction I can only visualize intense bands on an agarose gel for 2 of the 4 constructs (These 4 constructs only differ by 1 nucleotide).

Below I will describe the reaction I'm using and some of the ways I've tried to troubleshoot. I would GREATLY appreciate any additional suggestions or comments you may have:

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3ul 10x Pfu Buffer
1ul dNTP mix
1ul construct (50ng/ul)
1ul forward primer (30uM)
1ul reverse primer (30uM)
22ul water
1ul Pfu Turbo
------
30ul TOTAL

PCR Program
94 degrees for 2min
94 " for 1 min
51 " for 1 min
72 " for 3 min
30 cycles
72 degrees for 10 min
4 " forever

PCR Product Purification
Add 150ul PB (Qiagen)
Load on a Qiagen extraction kit spin column
Spin for 1min at 13000 rpm
Wash with 750ul PE (Qiagen) and 1 min spin
Remove flow-thru and 1 min spin
Add EB and elute DNA


So, all four constructs use the same primers, buffers, polymerase, etc. Troubleshooting myself I have found that the 2 constructs with only light bands work better at 57 degrees (step 3) and at 40 cycles instead of 30. In addition, I have also transformed some of my starting construct. These adjustments, however, do not help because following PCR purification I still have a digestion and gel extraction to complete. Following digestion and running the DNA on a 0.8% agarose gel, I cannot visualize any bands. The intensity of the bands (DNA concentration) for the other 2 constructs, however, was high enough to successfully complete a ligation.

What I'm trying to understand is: what is different about these two groups of plasmids and what can I do to fix my amplification dilemma?

Thank you for your help!
Nicole
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Old 07-21-2009, 01:11 AM
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Default Re: PCR troubleshooting for molecular cloning

Hmm..I am not good at this but I will try my best to give you some ideas.

You said that two constructs with only light bands work better at 57degcel and at 40 cycles instead of 30. By increasing the number of cycles, I would think that the PCR products will definitely increase, this provided that you have sufficient dNTPs and primers. However, nonspecific amplification can also occur with increasing cycles. One thing to note is that since you are using Pfuturbo, do you add the polymerase last? Because it has 3'->5' exonuclease activity, it can chew up your primers if you add it in advance of the rest of the components.

You can also try to find the best annealing temperature by doing a gradient PCR for your four constructs.
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Old 07-21-2009, 04:24 AM
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Default Re: PCR troubleshooting for molecular cloning

I have tried using a gradient, which made little difference. Also, I add Pfu turbo last, so I don't think that would play a role.
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Old 07-21-2009, 10:52 AM
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Default Re: PCR troubleshooting for molecular cloning

Do you run your PCR prior to PCR purification? You can lose quite a bit sometimes in that process. Also, I wouldn't be too comfortable using 40 cycles if I was trying to clone something.

Maybe try to get other polymerases to work, sometimes one just doesn't work, maybe add some DMSO to your PCR reaction?
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Old 07-21-2009, 11:40 AM
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Default Re: PCR troubleshooting for molecular cloning

just like to know
1. what is the size of ur construct/pcr product? according to ur extension time is ur construct over 1kb?
2. how much unit of pfu did u use?
3. ur primer anneal to the insert or the plasmid?

hope to hear from u soon
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  #6  
Old 07-21-2009, 01:38 PM
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Default Re: PCR troubleshooting for molecular cloning

Pigfarmer: I am using a purified construct. Also, I am not really comfortable with 40 cycles either, but the bands for all four constructs are clean and the correct size.
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Old 07-21-2009, 04:57 PM
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Default Re: PCR troubleshooting for molecular cloning

Butters:

1. PCR Product size is 2.5kb and my extension step is 3min.
2. 2.5 units/ul of Pfu
3. My primers anneal slightly upstream (was that the question you were asking?).

Also, does it really make sense for one nucleotide to completely alter the amplification of an insert, when the inserts are virtually identical?
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cloning , low product concentration , molecular , pcr , pfu turbo polymerase , troubleshooting


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