7 replies to this topic

[planktonica]

Hi there,

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!!!

Thanks

[perneseblue]

it should be noted that the columns do have a maximum binding capacity of 10ug of DNA.

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.

planktonica, on Mar 4 2009, 01:28 PM, said:

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?

[planktonica]

Uncut vector is initially 530 ng/uL, final vol: 36 ng/uL


I have around 500 uL of vector in total, but I use probably 10 or less for digestion.

[perneseblue]

sorry, I meant the total amount of DNA that have been digested.

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.

[NemomeN]

I agree with persen....the amount of dna you used is important...not necessarily the concentration.  Anyways, with gel extraction, you do lose on average about 50% of the DNA.
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.

[microgirl]

I always get very low yields with the Qiagen gel extraction kit - I've heard the Zymo kit is better. However, I've always successfully ligated my negative yields from the gel extraction with other negative yields and gotten plenty of colonies!

[planktonica]

Thank you so much for all your answers. Now I guess that the problem I have with cloning is not for insufficient vector. Still I find annoying getting low yields of vector, but it looks like the amount is enough.

[aimikins]

I have also seen very low yields with that kit. I typically do it old-school method: spin over glass wool and ETOH ppt. it's not quite as clean, but the yield is always better.

good luck with your cloning!