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Please please help me with my Phusion PCR.

Phusion PCR

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5 replies to this topic

#1 jerrywu1987

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Posted 04 March 2014 - 03:47 PM

I am trying to get a nearly 3kb fragment with Phusion (from NEB). The reaction was set up as follows:

1)      5X reaction buffer (I tried both HF and GC buffer) 10 µl,

2)      dNTP 1 µl (Final concentration 200 µM),

3)      Forward primer (designated F1) 1 µl (making final concentration as 1 µM),

4)      Reverse primer (designated R1) 1 µl (making final concentration as 1 µM),

5)      Template DNA (from cDNA) 2 µl

6)      DMSO 1.5 µl (making final concentration as 3%)

7)      Phusion Pol 0.5 µl

8)      Di-water up to 50 µl

 

The reaction condition is as follows:

Initial Denaturation  98°C (2min30s); 98°C (10s);  72°C (25s); 72°C (3min); Final Extension 72°C(10min)

35 cycles

 

I am sure my template does not have any problem, because I successfully got the correct fragments by using the primer F1 and another reverse primer R2 (about 1.2kb), as well as by using another forward primer F2 and the primer R1 (about 300bp). Also, the reason why I used 2min30s to initiate the denaturation is that I think my cDNA sample is a complex mixture. You may wondering why I use 72°C as the annealing temperature. Because I used the NEB Tm calculator online, and it gave me this answer. This annealing temperature also worked well with primer pair F1&R2.

The only different between the F1&R1 product and F1&R2 product is that the former is more than twice the size of latter. Why I already increased the extension time but was not able to get anything but some small size primer dimers. I check the primer dimer score of F1&R1 and it was not worse than F1&R2 and F2&R1. What happened to my Phusion PCR???

 

I appreciate your help!



#2 phage434

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Posted 04 March 2014 - 05:10 PM

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.



#3 perneseblue

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Posted 04 March 2014 - 08:14 PM

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.

 

i agree.  Only the part of the primer that actually anneals to the template is used to calculate the tm.


May your PCR products be long, your protocols short and your boss on holiday

#4 jerrywu1987

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Posted 04 March 2014 - 08:47 PM

I suspect that your annealing temperature is dramatically too high. You probably are calculating it based on the total primer length, whereas the important length is the portion which matches your template. I'd suggest an annealing temperature of 55 as a starting point.

 

Thanks for your answer. Both primers are 20~22 bp long, and all match my template. Moreover, I found the annealing temperature calculated by NEB Tm Calculator is also associated with the polymerase. If I use Taq, the annealing temperature will be 65C. But anyway, I will try temp gradient. starting from 55C. 



#5 phage434

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Posted 05 March 2014 - 05:20 AM

Such a high annealing temperature for a primer of 22 bases probably means your GC content is very high. Lots of problems occur with very high GC. You could raise the DMSO concentration to 6%, or (better in my experience)  switch from DMSO to a solution of 1 M Betaine. But I'm guessing that the gradient will fix things. This is another example of the near uselessness of Tm calculators, in my opinion. People actually believe the results, and they are rarely correct. Is there a decent GC percentage near the 3' end of the primer? High GC elsewhere and low GC at the 3' end will also often fail completely.



#6 vetticus3

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Posted 05 March 2014 - 07:35 AM

a normal phusion pcr reaction is this:

 

 

Initial Denaturation 98°C 30 seconds

 

25-35 Cycles

98°C - 5-10 seconds

45-72°C - 10-30 seconds

72°C - 15-30 seconds per kb

 

Final Extension 72°C 5-10 minutes

 

Hold 4-10°C  

 

generally, the 45-72°C step you'd begin with 55°C

 

what program did you use to design the primers?  something went very wrong somewhere.


Edited by vetticus3, 05 March 2014 - 07:37 AM.






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