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Agarose gel issue


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#1 netnus

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Posted 06 October 2004 - 04:12 PM

hi,everyone,

I run a agarose gel to see whether my insert has been ligated into the plasmid vector or not.
The ligation protocol is as following:
insert:vetor=2:1 (only estimation) total 20ul
T4 ligase buffer: 2ul
ligase : 3 unit
In addition to all above, I still add 0.5 ul (0.5M) ATP to make sure the ligase works.
After incubate at 22 degree for 1 hour, I run a gel. But instead of seperate bands, I see a smear in the lane where I load ligated solution.
Of course in this lane I still can see some relatively strong bands which belong to the cut insert and cut plasmid.
I feel really upset for I was stopped in the step of ligation for almost one month.
Hope I can get your advice soon!


netnus

#2 musicflower

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Posted 06 October 2004 - 05:54 PM

hi,everyone,

    I run a agarose gel to see whether my insert has been ligated into the plasmid vector or not.
  The ligation protocol is as following:
  insert:vetor=2:1 (only estimation) total 20ul
  T4 ligase buffer: 2ul
  ligase : 3 unit
  In addition to all above, I still add 0.5 ul (0.5M) ATP to make sure the ligase works.
  After incubate at 22 degree for 1 hour, I run a gel. But instead of seperate bands, I see a smear in the lane  where I load ligated solution.
Of course in this lane I still can see some relatively strong bands which belong to the cut insert and cut plasmid.
  I feel really upset for I was stopped in the step of ligation for almost one month.
  Hope I can get your advice soon!


netnus

why not transfer first,extract plasmid from bacteria ,then identify your insert cleaving with RE.

#3 netnus

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Posted 06 October 2004 - 06:16 PM

hi,everyone,

    I run a agarose gel to see whether my insert has been ligated into the plasmid vector or not.
The ligation protocol is as following:
insert:vetor=2:1 (only estimation) total 20ul
T4 ligase buffer: 2ul
ligase : 3 unit
In addition to all above, I still add 0.5 ul (0.5M) ATP to make sure the ligase works.
After incubate at 22 degree for 1 hour, I run a gel. But instead of seperate bands, I see a smear in the lane  where I load ligated solution.
Of course in this lane I still can see some relatively strong bands which belong to the cut insert and cut plasmid.
  I feel really upset for I was stopped in the step of ligation for almost one month.
  Hope I can get your advice soon!


netnus

why not transfer first,extract plasmid from bacteria ,then identify your insert cleaving with RE.

I tried transformation many times before but not even a colony grew in the LB-agar plate. So I don't want to waste any more cells strain before I can make sure the insert has been ligated.
My colleagues did the ligation before. They got obvious seperate bands which show the right size for the ligated plasmid.
Any other suggestions will be greatly appreciated!


netnus

#4 kant0008

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Posted 06 October 2004 - 11:27 PM

Hi,
try ligating overnight at 4C, or even over a couple of nights.
Also, ligase buffer should have ATP in it, you sure you're not overdoing it?
Also use no more than 1/10th of total volume of ligase, it's storage buffer may interfere (eg. only up to 2ul for 20ul reaction)
Good luck




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