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2 bands in blue colony PCR


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#1 bumbilko

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Posted 17 June 2011 - 02:24 PM

Hi,
I am doing PCR screening of my colonies pBluescript transformed in ElectroTen cells (Stratagen). I usually pick up 1-2 blue colonies as a negative control. I expect a product of about 250 bp, which are the bases between two M13 primers in an empty vector. However, I am getting two products: one of cca 250 bp a another one which is allways of about 2500bp long. Positive white colonies have only one band of expected length (more than 4 kb). I am using a very long 20 min extension step in my PCR. (Naturally, the 2500 bp band of a blue colony is not amplified with short extension steps, but I need long extension step to amplify my positive inserts.)
Why are there two bands in my blue colony PCR?
Thanks!

#2 Evanescence

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Posted 21 June 2011 - 12:09 AM

Hi,
My friend..may i know your pcr profile how long time u set for each...let try trouble shoot it...it always better if you give us complete info so we can get more hints :)

Keep trying will make u flying
Keep surrender will ensure "it's over"

soo keep try ok?:)

Evanescence

#3 bumbilko

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Posted 22 June 2011 - 03:30 AM

My PCR protocol is the following:

5 ul Accu Taq LA 10 x buffer
2.5 ul dNTP (10mM each)
9.6 ul forward M13(-41) primer 3.2 pmol/ul
9.6 ul reverse M13(-48) primer 3.2 pmol/ul
0.5 ul polymerase Accu Taq
21.8 ul water
1 ul 100 times diluted DNA sample from miniprep or directly picked colony

PCR steps:
98C 30 sec

94C 30sek
53C 30 sek
68C 20 min
30 times

68C10 min
8c forever

I strongly believe that the 2.5kb band in blue colony PCR is an empty plasmid which gets somehow amplified by PCR. Am I right?

Thank you very much for help!

#4 phage434

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Posted 22 June 2011 - 04:04 AM

Question 1: Why do you care? You can distinguish the correct vs. incorrect clones easily with your existing conditions.

If you care, then likely there is another binding location for one of your primers located 2.5 Kb away from the reverse primer. You could redesign your primers, or perhaps optimize the reaction to eliminate the binding with the existing primers. But why again do you care?

#5 bumbilko

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Posted 22 June 2011 - 04:49 AM

Question 1: Why do you care? You can distinguish the correct vs. incorrect clones easily with your existing conditions.

If you care, then likely there is another binding location for one of your primers located 2.5 Kb away from the reverse primer. You could redesign your primers, or perhaps optimize the reaction to eliminate the binding with the existing primers. But why again do you care?


Yes, you are right - I shouldnt care about this 2.5kb band at all. But this 2.5 kb band means that my PCR reaction might not be working correctly neither for my positive inserts. So I must optimize it... First thing that came into my mind was using shorter elongation step, but I cannot use it, because my positive inserts wont be amplified.

I just wanted to know if someone has had the same experience.

Thank you!

#6 phage434

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Posted 22 June 2011 - 07:24 AM

I would never trust a pcr band as a definitive proof of a construct in any case. You surely will go and sequence the correct clones. If you really really care, you can cut the 2.5 Kb band out of a gel and sequence it. Then you will know. And the answer won't be interesting, I don't think.




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