Posted 30 August 2010 - 07:14 AM
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.
Posted 30 August 2010 - 05:21 PM
Posted 30 August 2010 - 08:44 PM
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
Posted 31 August 2010 - 03:39 AM
Posted 01 September 2010 - 01:42 AM
thans homebrew and perneseblue!
Posted 01 September 2010 - 03:40 AM
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.