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
- - - - -

help


  • Please log in to reply
5 replies to this topic

#1 sofiap

sofiap

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 22 February 2010 - 11:17 AM

hello, I'm having some problems in getting colonies after transformation :rolleyes: . Never inactivate the T4 DNA ligase but always froze (-20 ° C) ligations before making the transformations. Is freezing sufficient to inactivate the T4 DNA ligase?
Thank you all.
Ps-I did ligations with molecular ratios 3:1 and 6:1 on a volume of 15uL and DNA concentration around 20ng/uL (in DH5alpha competent cells).
sofiap

#2 Cruise

Cruise

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 23 February 2010 - 07:52 AM

hello, I'm having some problems in getting colonies after transformation <_< . Never inactivate the T4 DNA ligase but always froze (-20 ° C) ligations before making the transformations. Is freezing sufficient to inactivate the T4 DNA ligase?
Thank you all.
Ps-I did ligations with molecular ratios 3:1 and 6:1 on a volume of 15uL and DNA concentration around 20ng/uL (in DH5alpha competent cells).
sofiap

/

1.Have the buffer you use thawed and mixed completely?
2.Have you put the ligation in proper temperature and for sufficient time? such as 16 degree O/N.
3.Are your competent cells efficiency high enough? Transform a plasmid as a postive control.

Hope those useful to you, good luck

#3 microgirl

microgirl

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 122 posts
0
Neutral

Posted 23 February 2010 - 08:14 AM

Maybe freezing is your problem? I have never even inactivated the T4, just gone ahead and transformed. Sometimes, though, what you're trying to transform in can be lethal to the cell so you don't get any colonies. Are you allowing them to recover in antibiotic-free media before you plate them? Are your positive controls growing?

#4 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
4
Neutral

Posted 23 February 2010 - 10:00 AM

I ligate at RT for 30min. and directly use it for transformation. The main thing, is to make sure the ligase buffer is not degraded.

#5 sofiap

sofiap

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 24 February 2010 - 07:56 AM

Hi, thanks you all for the tips.
1st- Yes i'm sure that buffer are completely thawed and mixed, although not completely sure if it is working as it should be (maybe this is the problem)
2nd- I did ligations at 16ēC O/N, O/W, 2h RT and results were always the same :)
3rd- my competent cells are recent and I tested the efficiency (about 8.9x10[sup]6 colonies /ug of DNA of plasmid) but I didn't positive control of my transformations (I do next time)
4th- Before plate, I put them in LB medium antibiotic-free for an hour with gentle agitation.
Next time I will do positive control and I'll try to check if buffer is not degraded.
Thanks
sofiap

#6 microgirl

microgirl

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 122 posts
0
Neutral

Posted 24 February 2010 - 08:02 AM

Bad ligase is almost always the problem with cloning in our lab. We had an undergrad spend 6 months trying to clone something only to discover that the ligase was bad. It is sometimes worth a call to the vendor to make sure your batch of ligase hasn't been "recalled."




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

©1999-2013 Protocol Online, All rights reserved.