Subcloning problem - (Jul/21/2008 )
I am having a problem that bogged me for 2 weeks. I am trying to create an expression vector by cloning a 2.7kb DNA fragments, extracted from a plasmid by double enzyme digestion, into a backbone plasmid of size 7kb, also digested with the same two restriction enzymes. this backbone plasmid is conferring Kanamycin resistance. the resultant plasmid was transformed into DH5alfa E.coli by hot shock and plated onto LB supplemented with 50ug/ml Kanamycin. Unfortunately, there was no colony. I have tried the experiment several times and I 'v got the results.
1-After double digestion, I extracted the desire fragments from gel. But after ligation I could not see any band.
2-procedures: double digest of about 500 ng of plasmids. purified by gel electrophoresis. elude in 10 ul and ligation of 3 ul of plasmid with 1ul of insert in a total of 10 ul . Transformation of 2 ~10 ul of ligation reaction into DH5alfa.
Any help would be appreciated.
Check the competency of your cells. It is suspicious that you got no colonies -- I would expect at least some background from your uncut or religated backbone plasmid.
You may also be damaging your DNA during gel visualization. Make sure you use long wave UV or blue light for transillumination and minimize UV exposure.
Your ligation buffer may also be dead. Again, a control reaction would tell you.
Thanx for the advice. I will check those points and tell the results.
I agree with phage about controls. I would add an extra one.
You say you gel purify but see no band after ligation. Do you check your purification by gel before ligation. I know is a pain, and I'm assuming you are using a kit and they are meant to work, but it might be worth running 1ul of your digested and purified insert and vector just to double check. This will also allow you to check quantities (roughly) to set up the ligations.
I had a similar problem recently where I was trying to cut out insert from a plasmid, gel purify and then clone and I could not get it to work.
So I used PCR to amplify up my insert ( primers flanking my digestion sites), digested, inactivated restriction enzyme, then treated with proteinase k and sds cleaned this up and hey presto my insert went in first time.
As a general rule I try to avoid gel purification if I can, some people seem fine with it but I think there is some voodoo involved!
I can post a more detailed method if you are interested
thanx for your cooperation. Actually, I purified the backbone vector and the insert by gel electrophoresis. Then the desired bands were extracted by kit. after extraction I run 2ul from each band to check the validity of gel extraction and it was perfect.
Yesterday, I check the competent cells and the medium and there was no problem in the both.
I really would like to amplify my fragment ( insert ) as stevo kindly suggested but I do not know if it is ok or not since the sequencing of the insert is important for me and I am afraid of some mutation during PCR.
Here I have a question. I know ethidium bromide would affect the DNA but do you think it is the origin of my problem knowing I run DNA in gel with ethidium bromide?
Thanx for u again and I will keep checking my experiment using more controls as you all suggested. More details are welcomed.
For years, we've added guanosine to agarose gels from which we're going to excise a fragment. It acts as a UV protectant (see here).
PCR is perfectly fine for cloning work- just use a high fidelity enzyme (such as my fav, Phusion from Finnzymes)
Still not working well!!!!!!!!!!!
Which enzymes are you using and for how long have you digested the vector and insert? Too long with some enzymes can hurt ligation.