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help with transformation! thanks!! - (Mar/26/2009 )

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Hello all, I have had trouble with this transformation for over a month now. I hope someone can help me! Thanks.

I have a 1.9kb insert, double-digested with XbaI and XhoI, that I want to transform into pFastBac vector (4.8kb), also double-digested with the same enzymes. I gel extract the double digest reactions with Qiagen kit, and have a concentration of about 15-20 ng/ul for the insert and somewhere 35-60 ng/ul for the vector. I'm not sure if these concentrations are important, but I do find that these concentrations work best for generating transformants. I've also had very low concentrations before, like 5 ng/ul for the insert, that do not work as well (not sure why, since the final volume is always 20 ul, so the final concentration of DNA should be the same...).

I use the NEB Quick Ligation kit, which is a 5min RT ligation. Then typical transformation protocol is used. The heat shock is usually 45 sec at 42o. Although I have also tried the 1 min at 37o. I usually do 1:1 and 1:3 vector:insert molar ratios, and also plate ligations for vector with no insert (negative control). Yesterday's plates had nothing on the negative control or the 1:3, and about 10 colonies on the 1:1. I screened a few of them, but they are all false positives. This has happened to me before with a different insert, where there was nothing on the negative control, but quite a few maybe 10-20 on the 1:1, and nothing on the 1:3. The 1:1 colonies were all false positives, so this trend is not a good sign.

There is a PstI site between the XbaI and XhoI sites in my vector, but my insert also has that, so I cannot digest vector without insert, without digesting everything. My colleague has suggested phosphatase, but I am not sure how this procedure works. Can anyone help??? Thanks!

-whatfangz-

Have you check your XbaI site for possible dam methylation?

You might want to increase the ligation time to 15mins.

You might also want to run some of the ligated DNA onto a gel to confirm that the DNA is ligating.

whatfangz on Mar 26 2009, 01:55 PM said:

There is a PstI site between the XbaI and XhoI sites in my vector, but my insert also has that, so I cannot digest vector without insert, without digesting everything. My colleague has suggested phosphatase, but I am not sure how this procedure works. Can anyone help??? Thanks!


The instructions manuals for using phophotase is found on company website that sells these ezymes. Ie NEB, fermentas.

But in general
1 Unit CIP will dephosphorylate 1 pmol of DNA in 60 minutes when in a volume of 50ul using NEB buffer 3 at 37 Celsius.

When using CIP beware that overdephosphorylation by CIP will damage the ends of the vector making it unligatable.

-perneseblue-

Thanks for responding. The XbaI site in pFastBac is followed by a PstI site, making the sequence GACG, rather than GATG, so it should not be methylated?

I will try the extended ligation time, and run some on a gel. Thanks for your suggestions!

-whatfangz-

I ran some of my old ligations on a gel, and they are all showing bands near the top of the gel. There is some smearing for the 1:3 ligation. I read about the bands at the top of the gel, and I suppose my ligation is forming concatemers? I'm not sure how to solve this problem.

-whatfangz-

Concatemers are formed if the ligase is too active or the ligation time is too long.

I always use bigger amounts of DNA for ligation than you (about 10-fold), so maybe your ligase makes concatemers because it hasn't got anything else to do?
Just a suggestion.

-mastermi-

mastermi on Mar 30 2009, 01:21 PM said:

Concatemers are formed if the ligase is too active or the ligation time is too long.

I always use bigger amounts of DNA for ligation than you (about 10-fold), so maybe your ligase makes concatemers because it hasn't got anything else to do?
Just a suggestion.



I use 50ng vector, and 17-54ng of insert (depending on 1:1 or 1:3, and the insert size). Do you use much more than this? And what ratios do you use? Thanks!

-whatfangz-

Do you now use vector:insert 1:3 or do you use more vector (50 ng comparde to 17 for insert)?

I usually use 100-250 ng vector and the threefold amount of insert (300-750). It's very important to have more insert than fragment, especially if you have to be aware of religation. But if you do a sticky end ligation and have cleaned your vector before, you shouldn't have problems with religation.
The difference is, that I don#t use a quick ligation kit. I ligate with "normal" ligase 1 h at RT for sticky ends and over night at 16C and with 10% PEG4000 for blunt ends.
But I think the problem with the concatemeres would be the same with both methods, just that the incubation times are different.

But maybe somebody who uses a quick ligation kit too can tell you how much vector and insert he uses...

-mastermi-

If I am doing a 1:1 molar ratio for vector:insert, I use 50ng vector and 17ng insert. For the 1:3, it is 50ng vector and about 54ng insert.

And concatemers, even if some are forming, shouldn't be the entire ligation product, I am guessing. Anyway, I will try with more vector and see how that works.



mastermi on Mar 30 2009, 02:14 PM said:

Do you now use vector:insert 1:3 or do you use more vector (50 ng comparde to 17 for insert)?

I usually use 100-250 ng vector and the threefold amount of insert (300-750). It's very important to have more insert than fragment, especially if you have to be aware of religation. But if you do a sticky end ligation and have cleaned your vector before, you shouldn't have problems with religation.
The difference is, that I don#t use a quick ligation kit. I ligate with "normal" ligase 1 h at RT for sticky ends and over night at 16C and with 10% PEG4000 for blunt ends.
But I think the problem with the concatemeres would be the same with both methods, just that the incubation times are different.

But maybe somebody who uses a quick ligation kit too can tell you how much vector and insert he uses...

-whatfangz-

I usually use vector:insert at a 1:5 ratio and use about 10x the concentration you do. I would consider digesting the DNA longer and increasing the ligation time. I have had success with overnight digests and ligations, but that was not with QuickLigase. Check the competant cell protocol also; cells thawed on ice, add 5-10ul DNA ligation, ice 30min then 30 second 42C heat shock is common followed by ~50ul LB or SOC medium addition at 37C for 1-3hrs, then spread ~50ul onto plate.
I would recommend phosphatase treatment of your vector only (do not do both insert AND vector!). It is a quick procedure. After RE digest, add phosphatase buffer and phosphatase enzyme directly to the RE reaction sample and incubate ~1hr at 37C, then separate vector fragment and insert on agarose gel. Gel purify and ligate as usual. The phosphatase treatment will prevent your vector from ligating back onto itself, though with 2 different cutters it is not as big of an issue than if you were using only 1 RE.

-NKatpase-

Have you checked the competency of your cells? Also, the "rapid ligation" is rapidly getting you nowhere. Have you tried a more traditional ligation? I know there's great faith in vector:insert ratios here, but over the course of twenty or so years, I don't think I've ever calculated a vector:insert ratio, and I have successfully produced hundreds of clones, using many different vectors and insert sizes -- one I recall was a 17 kb insert in pBlueScript.

-HomeBrew-
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