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Cloning <100 bp fragments into a plasmid


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#1 Jerry.Ahmed

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Posted 17 August 2010 - 04:45 PM

Hi Guys,

I have to clone an 85bp fragment and a 97bp fragment into a vector. I got these fragmens originally as single stranded oligos, so I annealed the complementary oligos and performed a double restriction digest so that the double-stranded fragments have the restriction sites that I need to perform the eventual ligation. Unfortunately, my ligation did not work. I used a 3:1 and 10:1 (insert:vector ratio) with vector+ligase (no insert) control and vector only (no ligase, no insert) control. When I transformed these into electrocompetent bugs, I only got colonies for the 3:1 reaction and nothing for the 10:1. Interestingly, colony morphology was very similar between the 3:1 reaction and vector+ligase (no insert) control. I understand that the fact that these two are similar could indicate that the vector is religating, but that seems unplausible as I had performed a double digest on the vector (using the same enzymes as I did for the oligos), run it on a gel and then cut/purify my fragment of interest. So unless there is some residual cut material, I fail to understand how the vector may religate with two different restriction sites on each end.

I have been at this for about a month now and have not been able to make any progress. If anybody can give me any suggestions/idea it will be much appreciated.

Thanks,
Jerry

#2 mickey

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Posted 17 August 2010 - 05:53 PM

Hi Guys,

I have to clone an 85bp fragment and a 97bp fragment into a vector. I got these fragmens originally as single stranded oligos, so I annealed the complementary oligos and performed a double restriction digest so that the double-stranded fragments have the restriction sites that I need to perform the eventual ligation. Unfortunately, my ligation did not work. I used a 3:1 and 10:1 (insert:vector ratio) with vector+ligase (no insert) control and vector only (no ligase, no insert) control. When I transformed these into electrocompetent bugs, I only got colonies for the 3:1 reaction and nothing for the 10:1. Interestingly, colony morphology was very similar between the 3:1 reaction and vector+ligase (no insert) control. I understand that the fact that these two are similar could indicate that the vector is religating, but that seems unplausible as I had performed a double digest on the vector (using the same enzymes as I did for the oligos), run it on a gel and then cut/purify my fragment of interest. So unless there is some residual cut material, I fail to understand how the vector may religate with two different restriction sites on each end.

I have been at this for about a month now and have not been able to make any progress. If anybody can give me any suggestions/idea it will be much appreciated.

Thanks,
Jerry



Have you ever done a miniprep to check the whether the insert is successful?

#3 Jerry.Ahmed

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Posted 17 August 2010 - 06:27 PM

Yes.. the miniprep does yield some DNA.. however when I do the check digest (using the same enzymes whose sites were initially used for ligation), I do not see my insert being released.All I see is that the enzyme have the vector, but nothing is released. So I'm assuming the ligation was not actually successful..

I have been reading other posts and a lot of people talk about phosphrylation of oligos and dephosphorylation of the vector. While I was performing this, I did not think about this.. Does that affect my reaction at all.

Thank you!

#4 Rosewater

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Posted 18 August 2010 - 03:58 PM


Hi Guys,

I have to clone an 85bp fragment and a 97bp fragment into a vector. I got these fragmens originally as single stranded oligos, so I annealed the complementary oligos and performed a double restriction digest so that the double-stranded fragments have the restriction sites that I need to perform the eventual ligation. Unfortunately, my ligation did not work. I used a 3:1 and 10:1 (insert:vector ratio) with vector+ligase (no insert) control and vector only (no ligase, no insert) control. When I transformed these into electrocompetent bugs, I only got colonies for the 3:1 reaction and nothing for the 10:1. Interestingly, colony morphology was very similar between the 3:1 reaction and vector+ligase (no insert) control. I understand that the fact that these two are similar could indicate that the vector is religating, but that seems unplausible as I had performed a double digest on the vector (using the same enzymes as I did for the oligos), run it on a gel and then cut/purify my fragment of interest. So unless there is some residual cut material, I fail to understand how the vector may religate with two different restriction sites on each end.

I have been at this for about a month now and have not been able to make any progress. If anybody can give me any suggestions/idea it will be much appreciated.

Thanks,
Jerry



Have you ever done a miniprep to check the whether the insert is successful?

Hi Jerry,
I don't think there should be any difference in colony morphology, unless the plasmid is affecting cell growth (besides just providing antibiotic resistance). Phosphatase treatment of the vector can certainly reduce background, but it is probably not necessary if your vector is double digested completely and gel purified. Your problem may be incomplete digestion of the insert and/or failure in the annealing of the oligos. I would have designed the oligos such that they have the desired overhangs, once annealed, so that you wouldn't have to digest them

#5 NemomeN007

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Posted 18 August 2010 - 05:36 PM

There is an assumption here that your enzymes are digesting your vector efficiently, which seems to not be the case. Furthermore, synthetic DNA fragments are not digested as efficiently either, so there is a double whammy of inefficiency which contributes to your difficulty. I would agree to anneal overhanging oligos to circumvent the need to digest, but would 5' kinase them as this increases the efficiency of sucessful ligation (reason unknown)

#6 fishdoc

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Posted 18 August 2010 - 05:57 PM

Yes.. the miniprep does yield some DNA.. however when I do the check digest (using the same enzymes whose sites were initially used for ligation), I do not see my insert being released.All I see is that the enzyme have the vector, but nothing is released. So I'm assuming the ligation was not actually successful..

I have been reading other posts and a lot of people talk about phosphrylation of oligos and dephosphorylation of the vector. While I was performing this, I did not think about this.. Does that affect my reaction at all.

Thank you!



Depending on the copy number and size of your vector, seeing a band less than 100 bp from a double digest could be quite difficult. There may not be enough DNA there to get a signal. You might be better off doing a single digest of your minipreps and compare it to a single digest of the uncut vector. If the vector is not too large,you should be able to see a difference of 100 bp with the right percentage gel.

Alternatively, use vector-specific primers outside the cloning site for PCR. An amplicon 100 bp larger than the one you get from the empty vector is the one you want. You can use minipreps if you want, but I find it's much quicker and easier to do colony PCR. You can screen a bunch of colonies that way, too.

#7 Jerry.Ahmed

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Posted 18 August 2010 - 08:26 PM

Hi Guys,

Thank you very much for your replies. Please allow me to fill in the blanks.

The size of the vector that I'm working with is 5.4kb and used 50ng of it for ligation in a 10ul reaction. My insert is 85bp and I used 2.36ng (3:1; insert:vector) and 7.86ng (10:1) of it. I only got colonies for my 3:1 ligation and these too looked very similar to the vector+ligase (no insert) control.

Another thing to note is that I did the double digest with KpnI and HindIII using NEB buffer2. HindIII works at 100% efficiency with NEB 2 but KpnI works only at 75%. So it may very well be that the digest is not complete. Unfortunately, I cannot use any other enzymes for the double digest as recognition sites from these enzymes are the only ones that have been added on to the ends of the insert.

Alternatively, as a lot of you have mentioned, I am thinking of designing primers specific to ends of my oligo and containing overhangs that correspond to the restriction site of the enzymes that I will be using to double digest my vector. I'll use these primers to PCR-up my insert with overhangs, clean it (via precipitation) and simply move on to the ligation step.

Does that sound plausible??

Thanks,
Jerry

#8 ElHo

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Posted 19 August 2010 - 05:14 AM

You also have to digest your pcr-product, which can be dificult. Otherwise you will end up with blunt ends for your insert. I would definitely recheck the clones you already obtained. If possible , try a double digest of your minipreps with one enzyme cutting your insert.




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