vector purification (for cloning)
Posted 04 March 2009 - 01:00 PM
I am using a 2.5 kb vector for cloning a 0.8 kb PCR product. After enzyme digestion with BamHI I proceed with the dephosphorylation step. After dephosphorylation I use Qiagen gel extraction kit (although I am not really extracting anything from gel) to purify my previously cut and dephosphorylated vector.
Initial vector concentration BEFORE digestion, dephosphorylation and purification: 530 ng/microliter
Final vector concentration AFTER digestion, dephosphorylation and purification: 36 ng/microliter
I tried several times and always I got around 30-40 ng/microliter...
Am I missing something?? I follow the protocol step by step, I even add 3M Sodium Acetate to acidify the sample and nothing seems to work. Either I am not getting rid of the alkaline phosphatase or I am somehow washing away all my DNA.
I need help!!!
Posted 04 March 2009 - 01:32 PM
Was enough QG buffer added to the solution? The QG buffer should be at least 3x the volume of the solution.
How about using phenol chloroform to remove the phosphotase? After the phenol-chloroform and ethanol precipitation, you need to wash the DNA pellet with 70% ethanol. Centrifuge the DNA sample before discarding the 70% ethanol wash to be certain that the DNA pellet has not dislodged from the side of the tube wall during the wash.
I add 5 times as much QG as my sample volume, as directed in the protocol. I really get low vector concentrations (around 10% recovery) and I am not sure if this is giving me problems with my ligation.
Very odd. How much DNA do you have in total?
Posted 04 March 2009 - 01:58 PM
I have around 500 uL of vector in total, but I use probably 10 or less for digestion.
Posted 04 March 2009 - 02:35 PM
30-40 ng/ul , in (perhaps) 50ul? In that is the case, I am guess there is 1.5ug of cut vector DNA. If the vector is only 2.5kb, that is enough vector for ligation.
Posted 04 March 2009 - 03:56 PM
When all the purification is done, I usually run 5ul on a gel and do a rough quantitation by comparing to markers. If the band(s) are very faint, the ligation will more often then not fail.
Posted 05 March 2009 - 07:26 AM
Posted 05 March 2009 - 09:12 AM
Posted 05 March 2009 - 10:00 AM
good luck with your cloning!