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Difficult Ligation - Details Inside

ligation

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9 replies to this topic

#1 RP2358

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Posted 13 January 2013 - 11:43 AM

Hey guys, so I'm having trouble with my latest ligation. My vector is 4448 bp and my insert is 296. My current attempt has 20 ng of vector and a 6:1 molar ratio insert:vector (resulting in about 8 ng insert) in a 10 ul reaction. 20 minutes at RT, 2 hours at 16C. Could the size difference be the problem? Should I go with a 3:1 ratio? 6:1 has always worked well for me, but I've tried this ligation three times. I have tried an overnight ligation. One point is that my insert is PCR product with cut sites less than 20 bp from the ends and the vector also has less than 20 bp between cut sites, so I've been gel-confirming but purifying the digestion directly (instead of gel purifying). I'd really appreciate any help with this. In case it's important, my insert is digested with XbaI+PstI-HF and my vector with SpeI-HF+PstI-HF in NEB Buffer 4.

Thanks!

Edited by RP2358, 13 January 2013 - 11:43 AM.


#2 Pangea

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Posted 13 January 2013 - 12:33 PM

XbaI is dam sensitive. Spe1 is compatible with Xba1. Maybe, use klenow and ligate blunt end. Did you do Re-cutting? PCR with Taq or Pfu? Generally, try different ratio for ligation.

Edited by Pangea, 13 January 2013 - 12:39 PM.


#3 RP2358

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Posted 13 January 2013 - 12:35 PM

They are isoschizomers. Thanks for replying though!

Edited by RP2358, 13 January 2013 - 12:35 PM.


#4 Pangea

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Posted 13 January 2013 - 12:41 PM

I already changes my mistake. Sorry.

#5 RP2358

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Posted 13 January 2013 - 12:50 PM

Maybe blunt...what do you mean re-cutting? And we do our PCR's with half Taq and half Vent. I may try a 3:1 ratio...

#6 Pangea

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Posted 13 January 2013 - 01:28 PM

Better to increase insert. On your Gel you dont see your expected size? Maybe difficult on this size of an insert. With recutting i mean where do you no that its not working?

#7 RP2358

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Posted 13 January 2013 - 01:31 PM

I do see the right sizes on the gel. And it's not working because I haven't been able to get any transformations. I guess technically that doesn't mean anything, but I find it hard to believe I would have a working ligation without transformations.

#8 Pangea

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Posted 13 January 2013 - 01:35 PM

I guess you use Calcium Chlorid and using all tranformed E. coli to plated on Amp plates.

#9 RP2358

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Posted 13 January 2013 - 01:37 PM

Huh?

#10 jerryshelly1

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Posted 15 January 2013 - 06:53 AM

Calcium chloride can increase the efficiency of a transformation, but depending on your e.coli cell type I would try throwing in some BME. When I was cloning a vector + insert around that relative size, the carbohydrates on the e.coli's outer surface were prohibiting proper entry. 2-4uL/100uL of cells should do the trick. have you tried confirming your insert post-ligation? Just try PCRing your vector + insert (find a primer within your insert + a primer outside, just to make sure you didn't ligate a doublet or triplet into your plasmid).

When you run a control with your cut plasmid, do you get any colonies? With your original plasmid? Check the efficiency of your e.coli cells with a stable plasmid.

Hope this helps





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