Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

No product after restriction digestion


  • Please log in to reply
26 replies to this topic

#1 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 08 June 2009 - 12:37 PM

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.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
405
Excellent

Posted 08 June 2009 - 05:05 PM

Purification didn't work?

#3 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 09 June 2009 - 12:24 AM

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

#4 epigenetics

epigenetics

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 68 posts
0
Neutral

Posted 09 June 2009 - 06:10 AM

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,

#5 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 09 June 2009 - 08:51 AM

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?

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,



#6 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 09 June 2009 - 08:53 AM

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

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



#7 medivh

medivh

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 09 June 2009 - 09:12 AM

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

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.

#8 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 09 June 2009 - 10:03 AM

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

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

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.



#9 mastermi

mastermi

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 82 posts
0
Neutral

Posted 09 June 2009 - 12:49 PM

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...

#10 jiajia1987

jiajia1987

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 212 posts
0
Neutral

Posted 09 June 2009 - 05:43 PM

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

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

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.

#11 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 09 June 2009 - 05:52 PM

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.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#12 Jaff

Jaff

    member

  • Active Members
  • Pip
  • 10 posts
0
Neutral

Posted 10 June 2009 - 02:17 AM

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!!

#13 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 10 June 2009 - 09:53 AM

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

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...



#14 xyz

xyz

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 10 June 2009 - 09:56 AM

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

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.



#15 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 10 June 2009 - 05:37 PM

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

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.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.