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PCR- 3.5KB region with added histag and restriction site


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#1 presidiogal

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Posted 28 April 2011 - 10:53 AM

I'm trying to amplify a 3.5 kb DNA region with GoTaq Polymerase.
I've added a histag and hindIII restriction on my reverse primer.

PCR
Cycle 1: 95 degrees for 15 mins
Cycle 2 (34x):
95 degrees for 45 sec
46-72 degrees for 45 sec
72 degrees for 3 min 30 sec
Cycle 3: 72 degrees for 10 mins
Cycle 4: 4 degrees

I've tried different annealing temperatures and also tried using 10% DMSO & 5% DMSO.
I know it's working because I'm running positive controls.

My forward primer is: ): 5- CTGGCCGGACGGTACTTC-3

My reverse primer is:

5-actAAGCTTTCAGTGGTGGTGGTGGTGGTGCCGGGCATCCGGCGAC-3
(The underlined region is the actual part that binds to my template and my HindIII site is bolded)
The GC content of my reverse primer is extremely high...~80%.
What should I do next?

Thank you so much!

#2 Adrian K

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Posted 28 April 2011 - 05:51 PM

Sorry, I don't understand.
You had design primers, you had done PCR, you confirm is working.
You dont seems suffer any problem do you? what you want to ask?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 presidiogal

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Posted 29 April 2011 - 12:09 PM

Sorry, I don't understand.
You had design primers, you had done PCR, you confirm is working.
You dont seems suffer any problem do you? what you want to ask?


My PCR is not working. (I'm assuming it might be due to my primers or annealing temperature)
I ran a positive control with a different set of primers , so I know my polymerase is working.

#4 Adrian K

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Posted 29 April 2011 - 02:46 PM

I see.. I thought you said your primers not working...
try use betaine solution, it "might" work, hopefully.
I will try not to design a primer with the his-tag there... what is the vector you are using? maybe we can start from there...

Alternatively, design a nested PCR: use your current forward primer, and your underlined reverse as first PCR. Clone the fragment into a vector, proceed to second PCR. For Second PCR, use your forward primer and the current reverse primer, maybe it might work this way if you cant change much on your vector.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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