cloning, got conoly, no dna at all. help! - (Nov/24/2006 )
i am doing cloning with two enzyme digestion, xho1 and not1 . vector is 10kb, and the insert is 2.7 kb. i am using the stratagen kit of ligation and XL10-gold competent cell.
i did 4 times. each time , i got around 5 colonies on the plates, but after mini, i got no DNA at all. i don't know what the problem. the colony grows well on the plate and in lb-ampisinin liquid. someone say maybe the plasmid go to the genomic DNA. but i don't know why in 4 times , it is always the case. i tried insert:vector ratio 3 to 1, 10 to 1 and 30 to 1.
so anybody has any suggestions?
thank you
jennifer
i did 4 times. each time , i got around 5 colonies on the plates, but after mini, i got no DNA at all. i don't know what the problem. the colony grows well on the plate and in lb-ampisinin liquid. someone say maybe the plasmid go to the genomic DNA. but i don't know why in 4 times , it is always the case. i tried insert:vector ratio 3 to 1, 10 to 1 and 30 to 1.
so anybody has any suggestions?
thank you
jennifer
How do you purify your plasmid DNA? Do you use a kit? In this case, maybe the columns cannot purify such a big plasmid
I agree with dnafactory, what are you using to purify the plasmid? If its a mini, maybe its just not enough culture column to purify the enough plasmid. And if its a column, perhaps the plasmid is too large to bind. Check the kit specs. Have you checked by PCR?
well put it simply... if you are using columns then 13kB plasmid is too large. The standard column will only bind efficienly DNA stands 10kB and below.
Old fashion alkaline lysis will work.
And while talking about this matter, what kind of plasmid do you have. Is it a high or low copy number plasmid? If it is low copy, you probably need to grow more cells
PS: Is it at all possible to screen by colony PCR? I am not sure how many colonies you are getting or how much DNA you us in the ligation reaction, but 5 colonies per plate sounds very low. It could well be false possitives.... bacteria which spotaneously aquire resistence to your antibiotic.
I don't think it is the problem of the column.
I have used Qiagen miniprep to purify my plasmids of about 14kb. I obtained quite high concentration of plasmid DNA.
But I have to emphasize that there are some modifications you need to perform, regarding the volume of buffers use in purification. You can find the modifications in the protocol provided with the kit.
Further, I used to preheat my elution buffer at 55oC.
Agree with virus fan, regularly perform "minipreps" with column based methods (qiagen miniprep) and get nice quantities of DNA.
I do mind the use of heated Tris for getting the DNA of the column, but apart from that use standard procedure.
Only thing that directly comes to mind: do you grow your bacteria long enough? The bigger the plasmid the longer it will take your bacteria to assemble enough of them to grow in the presence of your selective antibiotic.
I also agree that 5 colony's isn't much, but considering the size it might be OK. What about negative contol ligations, do they give any colony's?
Preheating the elution buffer is not stated in the protocol but the objective is to increase the amount of DNA yield. So far, I have no problem with it.
Yes, as vairus mentioned, the duration you grow your culture is also very important. Ideally, you should not grow it more than 16 hours.
Perhaps, you can grow up your bacteria in a 10mL culture medium for about 5 hours. Then, at the end of the day, you take about 100 microL of the bacteria culture from the 10mL culture and grow them up in the big flask of culture medium overnight (not more than 16 hours).
what is the strain of the E-coli into which you are electroporating your plasmid? are you sure it doesnt contain endonucleases. it was once my problem with no DNA from even conventional miniprep.
[quote]Endonuclease I Deficient: (endA) The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations. [quote] NEB website.
Old fashion alkaline lysis will work.
And while talking about this matter, what kind of plasmid do you have. Is it a high or low copy number plasmid? If it is low copy, you probably need to grow more cells
PS: Is it at all possible to screen by colony PCR? I am not sure how many colonies you are getting or how much DNA you us in the ligation reaction, but 5 colonies per plate sounds very low. It could well be false possitives.... bacteria which spotaneously aquire resistence to your antibiotic.
Yes. I think an alkaline lysis preparation is a good idea. That would be a good test for your miniprep kit because that is probably the issue. As for bacteria 'spontaneously acquiring resistance', seriously Perne you've got to be joking.
hi, it's me ,jennifer, i started the post. thank you for all your replies.
1. i tried the precipitation. i got very weak bands. so after cut, i can't say it's the right size or not
2. i tried to get the DNA with the column again, but i elute with 70 degree water. but i got nothing!
3. i tried to repeat the ligation and transfermation again, but since i run out of the competent cell from stratagen, i used the competetent cell from invitrogen. and i got no colony. i think the ligation and transfermation should be good. and the colonies i got should be the good ones. because my collage use the same ligase and competent cell from stratagen and has got the plasmid. also in his case , in ten colonies , he got no DNA from 9 , while from one , he got DNA, and it's the right one.
in my case, i am not so lucky.
any suggestions?
thank you
jennifer