6 replies to this topic

[ntlfly]

Hi all,
i am trying to ligate a 744bp PCR fragment into pet28c+ already containing my gene of interest (final vector size 7.1kb) with BglII/SalI enzymes.I am following NEB's recommendation to digest the PCR fragment no longer than an hr due to possible star activity of bglii.
I generally dephosphorylate the vector by incubating with CIP at 37 degrees for 1 hr,and eventually ppt the restriction digestions and do a gel purification the next day.
However upon ligation for 16 hrs, even upto 44 hrs at 16 degrees,i do not get any colonies upon transformation.I know transformation is working because my positive control is alright.I have done a ligation control to test the buffers and enzymes.When i run the ligation mix on a 0.7% agarose gel i see 2 distinct bands corresponding to the vector and the insert ,there is no evidence of concatamerization either,confirming that no ligation is taking place.
I would be immensely grateful for any advice.

Thanks so much
ntlfly

[mastermi]

Can you completely heat-inactivate your restriction enzymes?
Why do you make a gel extraction after phosphorolation? Agarose would inhibit the ligation reaction. Whenever it's not absolutely necessary, I prefer direct cleaning of DNA by ethanol-acetate precipitation (if necessary a phenol-chloroform extraction) or at least by a column before gel extraction.

Are SalI and BglI blunt-end cutters? If not: Do a sticky-end ligation for 1 hour at room temperature.

Edited by mastermi, 24 April 2009 - 11:06 PM.

[ntlfly]

mastermi, on Apr 25 2009, 02:05 AM, said:

Can you completely heat-inactivate your restriction enzymes?
Why do you make a gel extraction after phosphorolation? Agarose would inhibit the ligation reaction. Whenever it's not absolutely necessary, I prefer direct cleaning of DNA by ethanol-acetate precipitation (if necessary a phenol-chloroform extraction) or at least by a column before gel extraction.

Are SalI and BglI blunt-end cutters? If not: Do a sticky-end ligation for 1 hour at room temperature.

[ntlfly]

Hi Mastermi,
Thanks for your reply.
I cannot heat inactivate BglII.Both BglII and SalI both give sticky ends,so i am going to try out your advice for a 1 hr ligation at RT.
I do a phenol extraction in the method of gel purification, so there should not be any traces of agarose left to inhibit ligation,and it has always worked for me before.And besides,its also a double check for completely deactivating BglII.
I was wondering if i can completely leave out the CIP step.

Thanks,hope it'll work  

[T C]

Hey,

Precipitation does help...even if there is no agarose left, avoiding the gel specially for large fragments gives more colonies, n less junk.

You got to do it to believe it.

Best,
TC

ntlfly, on Apr 25 2009, 10:02 PM, said:

Hi Mastermi,
Thanks for your reply.
I cannot heat inactivate BglII.Both BglII and SalI both give sticky ends,so i am going to try out your advice for a 1 hr ligation at RT.
I do a phenol extraction in the method of gel purification, so there should not be any traces of agarose left to inhibit ligation,and it has always worked for me before.And besides,its also a double check for completely deactivating BglII.
I was wondering if i can completely leave out the CIP step.

Thanks,hope it'll work  

[hanming86]

After ur digestion ,issit possible to do a column clean up instead of gel extraction and try to work from that point on ward. I always try to avoid gel extraction whenever it's possible.

Normally ligation didn't work because of 1st agarose or 2nd high salt.

U could completely get rid of the agarose by passing the column with some volume of the gel solubilization buffer.

u could clean the product with EtOH precipitation .

[swanny]

I agree with hanming. A column purification, or even just EtOH precipitation will get rid of any RE hanging around.

You may have a bigger issue: PCR products don't cut well with SalI. Are there no other enzymes you can use? (Sorry, my knowledge of the pET28 map isn't complete!!)

Even if your SalI digestion is OK, you still don't know if  the ligase is working properly. Try religating the PCR product for ~20 minutes at RT; also ligate some DNA markers. Run both on a gel, as well as untreated ladder. Both samples should shift  significantly. If the insert only goes to the dimer-sized band, you know one of your enzymes isn't cutting: if so, I'd put money on the problem being SalI.