26 replies to this topic

[xyz]

Hi all,
I amplified my PCR product and it gave a nice band (4 microgram conc around), after that I puified it with qiagen kit and digested it with BAMh1 enzyme , after that I didn't get any band on gel. What could be the reason. I tried both  qiagen I and Qiaex II kit for purification but no yield. Need suggestions.

[bob1]

Purification didn't work?

[jiajia1987]

did u run a gel to check if you have your products after purification and before the restriction digestion?

[epigenetics]

Just one suggestion from my side.

As after purification with Qiagen kit, the concentration lowers , so check the concentration, run the gel. If you see the band, Then take appropriate amount to digest.
After digestion, sometimes instead of running agarose gel, for small DNA fragments run Native Page. It helped me a lot.

Thanks,

[xyz]

Hi
Thanks for suggestion. What is the rationale for native Page( poly acrylamide GE). and one more question is that checking the conc with spec (Nanodrop) makes any sense?

epigenetics, on Jun 9 2009, 06:10 AM, said:

Just one suggestion from my side.

As after purification with Qiagen kit, the concentration lowers , so check the concentration, run the gel. If you see the band, Then take appropriate amount to digest.
After digestion, sometimes instead of running agarose gel, for small DNA fragments run Native Page. It helped me a lot.

Thanks,

[xyz]

No I didnt do that. Just after purification digested the product

jiajia1987, on Jun 9 2009, 12:24 AM, said:

did u run a gel to check if you have your products after purification and before the restriction digestion?

[medivh]

xyz, on Jun 10 2009, 12:53 AM, said:

No I didnt do that. Just after purification digested the product

jiajia1987, on Jun 9 2009, 12:24 AM, said:

did u run a gel to check if you have your products after purification and before the restriction digestion?

you should have done that because it might save much time for you.
ps: bamhi has star activity from time to time at a high concentration.

[xyz]

Thanks.
Star activity depend on units of enzyme to conc of DNA , glycerol concentration and some other factors as well.I am using only 1 microliter of enzyme and that gives only 20units/microgram of DNA, so I am assuming that star activity is not giving problem to mu experiment.
If is there anything that I am not getting about star activity

medivh, on Jun 9 2009, 09:12 AM, said:

xyz, on Jun 10 2009, 12:53 AM, said:

No I didnt do that. Just after purification digested the product

jiajia1987, on Jun 9 2009, 12:24 AM, said:

did u run a gel to check if you have your products after purification and before the restriction digestion?

you should have done that because it might save much time for you.
ps: bamhi has star activity from time to time at a high concentration.

[mastermi]

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

[jiajia1987]

xyz, on Jun 10 2009, 02:03 AM, said:

Thanks.
Star activity depend on units of enzyme to conc of DNA , glycerol concentration and some other factors as well.I am using only 1 microliter of enzyme and that gives only 20units/microgram of DNA, so I am assuming that star activity is not giving problem to mu experiment.
If is there anything that I am not getting about star activity

medivh, on Jun 9 2009, 09:12 AM, said:

xyz, on Jun 10 2009, 12:53 AM, said:

No I didnt do that. Just after purification digested the product

jiajia1987, on Jun 9 2009, 12:24 AM, said:

did u run a gel to check if you have your products after purification and before the restriction digestion?

you should have done that because it might save much time for you.
ps: bamhi has star activity from time to time at a high concentration.

Sometimes, it is good not to assume. I use BamH1 in my work and it has a lot of problems (even if I see a clear and nice band after purification and after digestion).

And, mastermi is right, try to cloen it even if you don't see anything on the gel. I experienced this before and simply extracted the DNA from where I think the band would be (you know the size of your  insert) and cloned it and it worked.

[swanny]

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

[Jaff]

Thought for the day.................are you sure that your PCR product doesnt have any internal BamHI sites?  You may be purifying your product and (if you are going straight into your digestion step) may just be cleaving it to pieces!!

[xyz]

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

mastermi, on Jun 9 2009, 01:49 PM, said:

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

[xyz]

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

[swanny]

xyz, on Jun 11 2009, 03:56 AM, said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.