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Restriction Enzyme Inactivation & Pfu PCR


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#1 It'sNotMe

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Posted 11 October 2011 - 06:12 PM

Hi everybody,

Good day to all. I have three simple question that has been bugging me:

1. What are the effects on our digested product if RE heat inactivation , i.e. 65 oC, prolongs from recommended 10 mins, to 6 hrs?

2. Is Pfu polymerase amplification can be inconsistent with PCR condition setting? Because I amplified 1.6Kb product with the following -setting;

Initial denaturation: 95oC 4mins,
Denaturation: 95oC 2 mins,
Annealing 54oC 1 min,
Extension 68oC 1.5 mins (29 cycle) and
Final Extension 68oC for 10 mins.

It worked at first but failed on the subsequent attempts. I have triple checked all the PCR components and used new ones. But again, I was disappointed.

3. How do you check RE digestion of your end modified PCR product? Since only a few bp being cleaved of, I reckon you cant view them through gel electrophoresis. Is there any method to verify such end digestion?

Thank you for your time and attention. Hopefully someone can shed some light.

Best Regards,
It'sMe

#2 phage434

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Posted 13 October 2011 - 03:43 PM

If you really need to verify digestion, it can be done. Digest with one of the two enzymes in two reactions (one for each end). Then ligate the reaction with T4 DNA ligase (not the quick ligase version!). Heat kill the ligase for 20 minutes at 80C. Run the reaction on a gel, along with lanes of undigested and unligated controls. For the ligated reaction, you should see a double length product. Likely you will also see the single length product, but you can evaluate the efficiency of the digestion and ligation by comparing the intensity of the double and single length bands. I wouldn't want to have to do this every time I did a digestion, but it can help in debugging.

#3 It'sNotMe

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Posted 24 October 2011 - 04:05 AM

Thank you Mr./Mrs. phage434




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