2 replies to this topic

[mordiano]

Hi all,

Im using pBluescript SK vectors with my EST inserts.

My problem is that I have a huge diffrence of size between the sequences I have in the sequences (from the company that made the EST library and sequenced them) and what I see in agarose gels after cloning the inserts with M13R/F primers.

The bands in the agarose gels are always bigger by 400-800bps. The resolution of the agarose gels is indeed not that good (might differ 250 bases or so). The other reason Im thinking about is the fact that when the primers bind they actually carry over a bit of the plasmid into the PCR products.. but how much??

regards,

mordiano

[Warren]

mordiano, on Aug 28 2009, 09:02 AM, said:

Hi all,

Im using pBluescript SK vectors with my EST inserts.

My problem is that I have a huge diffrence of size between the sequences I have in the sequences (from the company that made the EST library and sequenced them) and what I see in agarose gels after cloning the inserts with M13R/F primers.

The bands in the agarose gels are always bigger by 400-800bps. The resolution of the agarose gels is indeed not that good (might differ 250 bases or so). The other reason Im thinking about is the fact that when the primers bind they actually carry over a bit of the plasmid into the PCR products.. but how much??

regards,

mordiano

Depending on which site(s) were used to do the original cloning, the M13 primers could be adding about 200 bp or so.  Warren..

[mordiano]

Hi!

I come to that conclusion when I look at the vector map

http://www.stratagen...pt_SK_minus.pdf

..observe that the sequence delineating the insert are bases 601-826 (ie 225 bases).


Does that make sense?

Regards,

mordiano