Gel extraction problem - (May/27/2005 )
I have tried doing a ligation using many different protocols and controls, but I still don't have colonies on my plates.
I finally came to the conclusion that after I extract my digested bands from the agarose gel, I lose my samples. I am using Qiagen gel extraction kit. For some reason, when I read the concentration, following extraction, in a spectrophotometer I am getting very low readings (which I believe is just background). When I proceed to ligation, there is no DNA in my tube and therefore I get no colonies.
I cannot understand why I am losing the DNA, since I have followed the Qiagen protocol exactly. I even checked the pH of my samples to make sure it is below 7.5, and it was around 6. I added more solubization buffer, but I still got a really low yield. I eluted with water and used a speed vac to try to concentrate my samples, but I still believe there is no DNA in my samples.
My vector is 5288 bp, digested with BamHI (single cut). My insert (734 bp) comes from a 1450 bp PCR product digested with BamHI (double cut). On the gel, these bands are in the right places and very clear.
My thoughts are that after digestion and dephosphorylation of vector, I should try to extract the fragments with phenol:chloroform rather than run on a gel (since I already know the sizes are correct). The digested PCR product will only contain one fragment with the correct complementary overhangs, so although I will have to screen more colonies, I will get enough DNA recovery to do a ligation.
Any thoughts suggestions or ideas?
It may not be a gel extraction problem. I've never had problem with qiagen gel extraction kit. You can run a gel using the extracted product to verify if the DNA is recovered.
I would run 5 ul PCR product after PCR and if there is only one single band, I would purify the product using the PCR purification kit without running a gel.
I commonly use Qiagen gel extraction kits and have had numerous problems. I find at best the recovery is at the lower end of expectation (20%), but are hesitant to move towards phenol/chloroform extractions.
The extraction efficiency is exceptionally bad from low DNA concentration to start with, if I have a strong band on the gel then I will recover upto 80% of the starting material. However, with a faint band at best 20% recovery.
This has meant that to ligate the faint bands I prepare multiple reactions giving a total volume of up to 200ul and then add my vector and ethanol precipitate the two together. I then resuspend my final DNA pellet in 10ul for the ligation.
Also I always run a check gel before ligating if I can see a band then it should ligate.
I have heard from a friend that even slight agarose contamination can inhibit ligation reaction. So his advice was to avoid gel extraction as much as possible.
I have the same problem of losing DNA after using QIAGEN's kit. Using the 30ul of elution buffer provided with the kit seemed to give me better results than with water.
I ran a small amount (about 50 ng) on a gel after I gel extracted and before I did the ligation. I saw a clear band for both insert and vector. I still got no colonies after ligation and transformation!
How about checking the competency of your cells?
if you see bands on a gel after the extraction then your technique there is ok.
Are you trying different ratios of vector to insert? I usually do 1:1, 1:3, 1:6 and 3:1 molar ratios (Promega website has lots on calculating these ratios).
As Kud0s says, it could also be the competent cells but a quick check with pure DNA would answer that question.
Hope this helps.
[quote=Roshni29,May 27 2005, 03:16 PM]
I am having the same problem with you. But I don't think that we have reasons for blaming the kit. I am trying to extract DNA from gel and ligate them into pGEM-T vector. the concentration of DNA is around 10-20ng/ul. this is engough for ligation, because the DNA frome gel will be used less for ligation than any other DNA ( that means that low insert : vector ratio should be better, I also have confirmed this point). Based on the control DNA transformation, I am sure that my competent cells I have made are excellent. I believe that in my experiment, some component from gel or electrophoresis buffer might inhibit ligation. One guy told me that sometimes, DNA from PCR can be ligated but not the DNA from gel. I am trying to confirm this now.
some points I can give you (I did not make sure if they are correct).
1) try to use TAE buffer, you can search by google, you will find that most protocol for extraction from gel will use TAE not TBE buffer.
2)try to add guansine(1mmol/L) in the electrophoresis buffer, why? please read the papaer: biotechniques,1996 vol21 No.5, 898
3) try to use UV-touch to view and cut the gel, finish the cutting in 45 sec
I am still confuesed that if the extension of time for disoliving gel by using the extraction kit will affect the DNA quality. sometime I have extend the time since the gel is not disolved easily.
I regularly use the Qiagen gel extraction kit for purifying my cut vector after phosphatase treatment and i never had any problem.
Things u can try:
Do heat treatment at 80 deg to inactivte bamH1 prior to addition of phosphatase.
U can add phosphatase directly to the cooled mix and incubate another 1-2 h at 37 and again heat inactivate by heating at 70-80 for 10-20min.
Load it onto a gel and do the gel extraction.
do additional washes with the QB yellow buffer to remove excess agarose and also do and addition wash with the PE buffer.
(Check the buffers, did u add ethanol to the PE buffer prior to use, i made that mistake once, and my dna was washed off )
Elute with either water ot EB, but do an ethanol precipitation to clean and concentrate the vector.
You can do the same treatment with insert except the dephosphorlytion step.
and set up a ligation with 100ng vector and insert in ratio 1:3 which in ur case should be 42ng , in a 10 uL reaction
I have also faced the same problem earlier. You can reduce the gel extraction to minimum possible times. Only once after restriction digestion should be enough. After dephosphorylation Phenol:Chloroform extraction is enough. Check pH of Phenol and Na Acetate. Or best is start with more DNA(Bulk digesting ~10ug DNA)!!