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Subcloning - RE digest and ligation and transformation


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#1 rainnie

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Posted 28 August 2012 - 06:58 PM

Hello, I've some question regarding my subcloning experiment.

I was trying to excise an insert from vector pCMV-SPORT6 and subclone the insert into vector pNEB193 (2.7kbp). The pCMV-insert was already provided for me.



First, i used Xbal (sticky end) and EcoRV (blunt end) to digest my recombinant plasmid (pCMV-SPORT-INSERT), which excised my insert (3.8 kbp) from the vector pCMV. After restriction digest, i analyse the digests on gel and i obtain the expected banding patterns corresponding to my insert (3.8 kbp) and vector (4.4kbp).

I digest my next vector pNEB193 with pMEI (blunt end) and Xbal (sticky end) and run my digest on the gel. Once again, i obtain a fragment the size of ~2.7 kbp, which correspond to the expected size of the vector.

I used Qiagen Gel extraction kit to extract my insert and vector pNEB from the gel and then run overnight ligation using NEB T4 ligase and its buffer.

Next day, i used calcium chloride to make my E.coli DH5 alpha competent and then transform the cells with my overnight ligation. The next day, i found that i have approximately 50 colonies on the LB-ampicillin plate.


The problem is: When i extract the plasmids from several clones and restrict digest them with Xbal and HindIII (this will excise the insert again from pNEB so that i can subclone it into my next vector) and then run them on the gel, the banding pattern doesn't show my insert. The expected banding pattern would be my insert (3.8kbp) and vector (2.7kbp). But mostly, i observed a band the size of approximately 2.7 kbp, which correspond to the size of my vector pNEB alone.


Can anyone tell me the reason for this? I thought that re-ligation of the vector is impossible since the ends are incompatible?

Also, could it be possible that the cells are transformed with linear vector pNEB?

I was told that re-ligation could be possible as bases can sometimes be lost at the ends?


A little help explaining this would be really appreciated, since I'm doing a write-up on it and I'm kind of stumped.

#2 bob1

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Posted 28 August 2012 - 07:37 PM

Blunt end ligations are very inefficient, it is quite likely that the insert isn't in there. How did your controls look? Did you do any controls to confirm that the restriction digests were working properly?

#3 ascacioc

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Posted 28 August 2012 - 10:48 PM

Re-ligation happens because of only one cut by one RE in the vector while the other site remains intact --> compatible ends. When ligase is applied, you get the compatible ends re-ligated in the original vector. There are 3 ways you can avoid religation (or at least reduce it)
1) dephosphorylate your vector; T4 DNA ligase needs phosphate at the 5' end to do its job so the ends of the vector are not compatible with each other
2) after ligation, you can use a 3rd RE that is in the MCS of the vector (in between your other two REs used for subcloning) but not in your insert; this will relinearize your religated vector making not transformable
3) use a vector that has a ccdB (lethal gene in DH5alpha; needs a special strain for the vector carrying it to survive --> kills off relegations) or sacB (kills the bacteria growing on sucrose); but I think that the flexible choice of vectors is not an option; I know that these kind of vectors were used by my supervisor in my master thesis when he wanted to clone with blunt ends. I personally never use blunt ends because they are a pain in the ... I mean out of all REs that are out there, there are more sticky ends ones than blunt ends...

Andreea

#4 rainnie

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Posted 29 August 2012 - 07:50 AM

Thelomitra pulchella@ I suspect my insert isn't there too, as the band on my gel only correspond to that of my vector.
I know that blunt ends are harder to ligate and transform. But since the other end (XbaI) is a sticky end, wouldn't that mean that the the recombinant construct would end up as a linear construct, assuming that the sticky end ligates but the blunt end fail to? as for my HindIII/Xbal digest, I'm sure it's working since i did use the enzymes to digest my vector (pNEB) alone as control.

Enthusiast@ Thanks for the suggestions. Should i dephosphorylate my vector right after restriction digest? As in, before i do the gel analysis anhd gel extraction?

Thanks for the help!!

#5 ascacioc

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Posted 29 August 2012 - 11:17 AM

http://www.neb.com/n...roductM0289.asp Read here about it. Last time I did a dephosphorylation for cloning, I was a master student :) I always skip this step and hope for the best from my colony PCR. But I also chose always sticky ends.

What I want to say is that I might be wrong when I state: dephosphorylate after gel extraction that you perform after restriction digest because you want to change the buffer into the phosphatase buffer and it is easier if you elute your DNA into water and add 10X phosphatase buffer+ phosphatase. But this makes the most sense to me right now.

Andreea

#6 metionina

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Posted 05 September 2012 - 02:47 PM

I did several ligations as yours: sticky + blunt ends.
1. keep your ligation volume as small as you can
2. what's your vector-insert ratio? Try different of them: 1:1, 1:3, 1:5
3. adding NaCl and PEG in your ligation buffer can help blunt ended ligation (in a volume of 20-35 μl I used 1.5 μl NaCl 2 M and 1 μl PEG 4K)
4. yes, dephosphorylation can be done after digestion and gel extraction. But it didn't work fine for my experiments
5. few colonies are good, so, analyze many of them




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