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
- - - - -

Need help for restriction enzymes


  • Please log in to reply
5 replies to this topic

#1 chat65

chat65

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 27 October 2009 - 09:53 AM

Hi
I got the problems about digest my insert and vector by restriction enzymes. I use BamHI and SalI to digest my vector (pET duet1) and insert genes (double digestion). Then Incubate at 37C for 2 hrs. Is that enough for 2 hrs?. After that I did ligation. Transformation and I got some colonies. Next step I did PCR colony screening but I did not find my expected band on the gel. I think it was because of restriction enzymes did not work properly. So any idea that I can confirm my restriction enzymes work? Please help.

#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 27 October 2009 - 12:32 PM

Did you gel-purify your digestions before ligation?

Can you recover plasmid DNA from some of your transformants and digest it, looking for an insert?

Perhaps your PCR colony screening failed -- did you have a postive control?

#3 chat65

chat65

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 27 October 2009 - 01:05 PM

Did you gel-purify your digestions before ligation?

Can you recover plasmid DNA from some of your transformants and digest it, looking for an insert?

Perhaps your PCR colony screening failed -- did you have a postive control?


Thank you for your advice
Yes I did purify my digestion before ligation.
I got colonies and I checked PCR colony screening and I got band but it was not the right size about 900 bps. I found band which lower than my expected size. It was false positive one I supposed to say. I don't know what should I do next. Any idea for me please

#4 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 27 October 2009 - 06:28 PM

If your insert does not have an EcoRI or BspMI site, you can do the ligation (but I'd suggest you do 2 hr @ RT, not 37) then digest with the enzyme; this will linearise any single-cut plasmids, but not affect your double-cut, insert ligated plasmids.

To check the REs, just do single digestions and run on a gel. To test the ligase is working and the insert is correctly cut , treat some DNA ladder and some insert and run on agarose - the ladder should shift up and the insert should have at least a trimer-sized band.

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.
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.

#5 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 27 October 2009 - 06:34 PM

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.


I completely agree. I used to just pick some small amount of a colony into 30 ul of PCR master mix, but then switched to picking about the same amount of colony into 50 ul of sterile water and using 1 ul of this suspension as template. My success rate went up dramatically.

#6 chat65

chat65

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 28 October 2009 - 05:20 AM

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.


I completely agree. I used to just pick some small amount of a colony into 30 ul of PCR master mix, but then switched to picking about the same amount of colony into 50 ul of sterile water and using 1 ul of this suspension as template. My success rate went up dramatically.


Thank you guys I will try and let you know later
Cheers




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

©1999-2013 Protocol Online, All rights reserved.