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TA cloning kit problem - No single colonies after transformation!!! (Sep/08/2004 )

Dear all,

I'm trying to clone a 2kb insert using Promega pGEM-T easy vector cloning system. I've tried different molar ratio for insert and vector in ligation, but all gave totally negative result--not a single colony can be seen!!!! I've followed strictly the protocol provided and i'm quite sure that the kit should be working fine as my labmate who is also using this kit gave positive result... i'm wondering whether it's difficult to clone a 2kb insert into a 3kb vector??!! Previously i've trying blunt end cloning but it also gave blue colonies only (i'm using pBluescript as vector and SmaI digested...)
The followings are all the possible problems i can think of:

1) My insert was PCR product and i've gel extracted it and eluted in 50ul water.... then i've tried to add A tail to the product as i'm afraid the A end added by Taq during PCR may be lost cus i've left the products for several days before i proceed to ligation..... So after adding A tail, i used PCR purification kit to purify it and also elute in 50ul water.... i then used 1ul of the eluate to run gel in order to check the concentration... i barely observe a weak band and so i assumed that 1ul of the DNA would contain ~10ng. After that, i speed vac the DNA to 3ul and proceed to ligation using TA cloning kit. i've used 25ng vector only. The ligation was done at 4oC overnight.... Would the speed vac damage the DNA and cause a problem in ligation?? i've speed vac for almost 1 hour.....

2) What should be the probable molar ratio for insert to vector?? Should i use more insert like 10:1?? Would too much insert cause a problem?? last time i've added many insert and the molar ratio should be around 20:1... and nothing can be seen after transformation... So could it be the problem of excessive insert?? But even if the insert will ligate with themselves (tho' i dun think it's possible cus they all have imcompatible A ends), i'm sure some insert can ligate with the vector.....

3) How do u guys determine the concentration of purified dna product?? i've tried spectrophotometer and also visualization thr. gel photo... The former gave nearly no absorbance (i've used 995ul water and 5ul DNA)... And for gel photo, i was told that a weakly observable band should contribute 10ng DNA... but did the brightness of the band affected by the size of DNA also?? i.e. if my DNA is 2kb, it will give same brightness as a 500bp band even if it has a lower concentration??

Thank you for spending time to read such a long passage.... your help is highly appreciated!!!

Thanks so much^^

-shicaeon-

hi shicaeon!

I'll try and answer some of your questions:

1)in my experience the A-tail is there or not, if you use Taq, then it's there and stays there a looong time - i once cloned a PCR product several weeks old successfully! So no need to add a A and purify again - this will result in loss of PCR product only! But maybe the "speedvacing" can be a problem since you concentrate not only DNA but everything else also (e.g. salt, enzyme etc.). I usually found to go with the manufacturers protocol gives best results (i.e.: PCR with Taq, then use 5Ál PCR Product and 1 Ál Salt, Ligate, Transform - There we are!)

2) The ususally given optimal MOLAR ration between insert and vector is 3:1 (Sambrook et al). It's known that higher ratios (10 or 20:1) can inhibit ligation. BTW, you did check your competent cells for viability and competence, did you? (Positve Control)

3)PCR products give in my experience almost no extinction at 260. So I always estimated from the agarose gel / EtBr stain with some other DNA of roughly the same size and known concentration. The "factor" I used was, that a weak band would be about 20ng/lane. And yes, not only the concentration of your DNA affects how much EtBr is there in you band (i.e. how bright the band is), but also the length of the DNA (longer DNA, more molecules of EtBr will intercalate in ONE DNA molecule).

mike

-jadefalcon-

I agree, don't worry about the A, just purify the PCR product by gel extraction or any PCR cleanup kit.
For the ligation, try using 50ng of vector with the proper molar ratio of insert (don't speed vac...the 10ng/ul is fine). And always do the positive control in transformations, along with a negative control for your ligation: vector only, no insert, it will save you a lot of headaches!!)
Also I've had situations where I had an insert, confirmed by a restriction digest for a site not present in the vector, but the colonies were still blue...if it's in frame there are instances where you won't see a color change. I doubt this is the case...since your insert is large, but just in case check one or two of your blue colonies.
And yes I believe larger bands are brighter because more EtBr can intercalate into the fragment of DNA.
I think SmaI has methylation problems...if you grew pBluescript yourself...you might want to double check on that...

-phegene-

I agree to phegene.

Blue/white screening never worked properly for me - i had empty white and successful blue clones. So nobody around here relies to that anymore. And when you're desperate you'll pick every colony, regardless if it's blue or white...

mike

-jadefalcon-

I was just wondering if you made sure that your PCR primers did not have a 5'T, because Taq polymerase is less efficient at adding a 3'A next to another A.
I once inadvertently used a primer containing a 5'T and got very few colonies. I redesigned the primer and the new primer had a 5'G. This time, I got more than 100 colonies.

-slab-