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cloning problem


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#1 andrcc

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Posted 30 August 2010 - 07:14 AM

Hi all,

I'm trying to clone a number of genes. Size range from a few hundred bp up to 2kb.
PCR amplified by pfu, purified. prety good yeild.
vector digested by SmaI, CIP-ed and gel purified. I'm just using pBluescript, coz our lab's expression vector always give poor sequencing data.

the ligation appear OK. i got quite a few colonies. PCR checked. strong band with right size.
i did preserve the clone for PCR by streak it on another fresh plate (with proper antibiotics), but the problem is: when i picked the clone for sequencing. the sequence is just nothing like my desired sequence. i cant even find my PCR primer sequence.

what's wrong? any comment would be much appreciated.

thans!

#2 HomeBrew

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Posted 30 August 2010 - 05:21 PM

Did you patch the putative transformants to a fresh plate before checking them for the insert by PCR, or did you do the PCR right from the colonies on the transformation plate? I find that I get a lot of false positive PCRs if I try and screen the colonies right from the transformation plate. I always pick a few dozen transformants to a fresh plate, grow this overnight, and PCR from these to check for an insert -- this cuts way down on false positives at the screening stage.

#3 perneseblue

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Posted 30 August 2010 - 08:44 PM

Did your colony PCR amplify the junction between DNA fragments? Only the junction between DNA fragments is unique to a ligated plasmid. Ie you are PCR amplifying across the junction between adjacent DNA fragments.

PCR is a very sensitive. It is sensitive enough to amplify naked DNA sitting on the agar plate. Thus the left over DNA from the ligation mix, now sitting on the plate can act as a template and thus give rise to a false positive signal if you PCR the insert. The same problem will not be seen if you amplified across the junction between DNA fragments
May your PCR products be long, your protocols short and your boss on holiday

#4 HomeBrew

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Posted 31 August 2010 - 03:39 AM

perneseblue is correct, I should have said "If my screening primers are both internal to my insert, I always pick a few dozen transformants to a fresh plate...".

#5 andrcc

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Posted 01 September 2010 - 01:42 AM

i see, i think that very likely to be the problems. coz im using the same primer for gene amplification and screening on the transformation plate.

thans homebrew and perneseblue!

#6 HomeBrew

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Posted 01 September 2010 - 03:40 AM

Another thing most of us have found that cuts down dramatically on false *negatives* when doing colony PCR is to first swirl a small amount of your colony (about the amount you'd normally would use for colony PCR) into 50 ul of sterile water, and then use 1 ul of that suspension as template.

The combination of first picking your transformed colonies to a fresh plate (to reduce false positives) and resuspending a bit of these colonies in water before use as template (to reduce false negatives) is very helpful in getting accurate colony PCR results the first time, and actually reduces the time spent and consumables used in the experiment by avoiding having to repeat steps due to ambiguous or incorrect results.




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