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PCR and then ligation


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#1 serundipity2009

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Posted 14 November 2009 - 08:11 AM

Hello,
I am doing PCR to amplify a 700bp region with primers with Tm= 81 and tm= 70 C. Would an annealing temperature of 65 work?

#2 noelmathur

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Posted 14 November 2009 - 10:12 AM

I am still wondering why would you dsign primers with such high Tm (I would like to know) I would go bit more, around 69C. The best option is run a gradient with 10 microliter PCR reaction to see what will work.

#3 serundipity2009

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Posted 14 November 2009 - 10:43 AM

I am still wondering why would you dsign primers with such high Tm (I would like to know) I would go bit more, around 69C. The best option is run a gradient with 10 microliter PCR reaction to see what will work.


My primer has a high melting temp because i need to amplify a ORF and the suggested bp to include is 18-22 and i also need to add 10 his and a restriction site for insertion into another vector. I have tried using a gradient centered at 65 C with 5 reactions. So the annealing temp was at 55, 60, 65, 70, and 75. The result: no amplication of my desired 700bp fragment. I only saw primer dimers :lol:

I used 2 ul of 10uM Primers. Should i decrease the primer added to either 1.5ul or 1ul? Should i increase template DNA?
Another reason that the pcr failed might be that i mixed all the pcr reagents the day before and kept them at -20 and then the next day, added the DNA template. Will this have an effect?

Thanks :D

#4 HomeBrew

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Posted 14 November 2009 - 12:17 PM

Do not include the 5' additions in calculating your primer Tms. What are the Tms of the primer segments (presumably 18 - 22 bp) that are homologous with your template?

#5 serundipity2009

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Posted 14 November 2009 - 03:21 PM

Thanks HomeBrew. Without the 5' additions, the Tm is 75 C. Why are the 5' additions not included?

#6 HomeBrew

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Posted 14 November 2009 - 07:13 PM

Thanks HomeBrew. Without the 5' additions, the Tm is 75 C. Why are the 5' additions not included?


Since they don't match your template, they don't anneal, so they don't melt either. Removing 10 his (30 bases) and a restriction site (6 bases) only brought your Tm down to 75 (from 81, I presume)? Your other primer was 70, and now it too is 75? I'm confused...?

#7 noelmathur

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Posted 18 November 2009 - 01:18 PM

Homebrew is right. Only calculate Tm for the sequence that is 'actually' binding. You are 'creating' a HIS-tag and a restriction site. Now taking into scenario that your primer is of 22bp, you have to have only G and Cs in your primer for Tm to be 70C. Please recheck.

Only issue with product not getting is usually annealing temperature. Read your polymerase manual and run a gradient -5Tm to +5Tm of your low Tm primer among both and see whats best temp to run a PCR so that you get your desired product.

#8 piemmea

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Posted 18 November 2009 - 02:19 PM

Homebrew is right. Only calculate Tm for the sequence that is 'actually' binding. You are 'creating' a HIS-tag and a restriction site. Now taking into scenario that your primer is of 22bp, you have to have only G and Cs in your primer for Tm to be 70C. Please recheck.

Only issue with product not getting is usually annealing temperature. Read your polymerase manual and run a gradient -5Tm to +5Tm of your low Tm primer among both and see whats best temp to run a PCR so that you get your desired product.


I agree. I would also suggest to run 10 cycles using the Tm of the binding part of your primers, then you can increase the Tannealing, considering that after those cycles the whole primer will then anneal




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