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Losing insert from pGEM?


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#1 Vegemite_Warrior

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Posted 17 April 2009 - 11:21 PM

Hi

i have recently been looking at presenilin levels as a function of alzheimers pathogenesis for my final year project which involves lots of RT-PCR!

i have made my own standard by subcloning a region of the presenilin gene (about 500 kb) from zebrafish into pGEM to create my taqman standard curve,i am using other peoples primers and probes which have been working fine for them.

my standards were working fine for about a month then all of a sudden it went wrong somehow,

my question is: is it possible for an insert to be lost from pGEM ?


any thoughts ? am i stupid for thinking this ? is there another explanation ?

Ta

#2 Carlton H

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Posted 20 April 2009 - 03:55 PM

Presuming that you were creating plates from multiple colonies, then no, you shouldn't be losing your insert, since that would be a fairly rare occurrence and it happening multiple times to different colonies would make it a very rare occurrence.

This is going to sound like a dumb question, but are you sure your colonies are still okay? I only ask because the cloning worked initially.

-Carlton
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#3 Vegemite_Warrior

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Posted 22 April 2009 - 04:36 AM

Hi

i have been done 10 colony pcr's from insert containing clones and negative control clones.
Suprise suprise the pcr's were successful from the insert containing colonies and not from the other ones.

A post-doc in my lab suggested that the T-A boundary that you clone into on pGEM is unstable and that at any point you could lose your insert (!?!) i asked for a bit of an explanation but was fobbed off !

i didnt think that an insert could be lost from a plasmid once stuck in with ligase

is this right ? (i have only been doing this for a few months not a few years like my post-doc!)

thanks for your help

V_W




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