my experience with cloning - (Nov/03/2006 )
I just want share my experience in cloning cDNA into plasmid because I spend a long time on it and find out the reason which is rare but important. it might be helpful to you.
In brief, i did everything correct from double digest of plasmid, PCR cDNA, PCR product cleaning, and double digest PCR product, ligation reaction. But the reason I could get not single clone is
1. you absolutely need to use buffer QG to wash after DNA binding to the column. The kit i used is Qiagen gel extraction, and from the instruction mannual, it said not necessary unless you are doing highly hygine steps such as microinjection. No need for regular ligation. NO!!!If you don't do this, you get no clones at all even ligate a single cut plasmid itself!!! really misleading!! because if it's really not that important i should not get not a single clone for single cut ligation of the plasmid!
2. I used NheI and XhoI for double digest. I found out many times that although the cleavage effciency is almost the same, the ligaiton effieicency is 10 times difference. SIngle cut by XhoI after ligation can get 1000 clones(using a lot for ligaiton and transform), while under the same condition, NheI get 100. I did a parrallel experemints using a smalled amount to ligate and transform, found out that XhoI cut ligation get 100 clones while NheI get 10 clones. So that's why when I do the double digest of the plasmid and the PCR product and ligate them I get only TWO!!! clones. But luckily, they are both posotive clones after I sequence them!!
Hope this might help you. And thanks again for many people help me when I post my problems on.
"I did a parrallel experemints using a smalled amount to ligate and transform, found out that XhoI cut ligation get 100 clones while NheI get 10 clones. So that's why when I do the double digest of the plasmid and the PCR product and ligate them I get only TWO!!! clones. But luckily, they are both posotive clones after I sequence them!!"
NheI cuts about 1/10th efficiency for supercoil DNA, so this may explain part of your observation. The second part of your statement is false math. You can not come to that conclusion from the data you have.
The first part about the Qiagen Gel also does not necessarily make sense. Are you cleaning up a pcr product directly or are you using a gel fragment or DNA straight after a digestion?
I cleanup the PCR product using PCR purification kit and after enzyme cleavage cleanup by gel extraction. my college did the same experiment, using all my reagent and everything is the same except that he use buffer QG to wash again. So i set up my experiments with everything the same just one with washing the other not. The result comes out always washing grow and no grow without washing(I try this may times on single cut of different enzymes)! I believe this because I checked the enzyme activity, different time-length cleavge, sequencing cut or double cut, running gel to make sure after purify by kit it's still there... There's not problem with my procedures, the washing is the only difference! I cannot see any other reason that should comes out always not a single clone growing on the plate for even single cut ligation product! I don't know what's your opinion on that?
" NheI cuts 1/10th efficiency of supercoil DNA"
This is where the problem is. If the cutting efficiency is so low, that means when you have your plasmid cut by NheI, say >50% of your plasmid is not cut, they remained supercoiled, you run the gel you should see apparently two bands, one faster the other at the right size position. I always see one clear band. Also, even if the difference of the supercoiled and linearized are not easy to tell and maybe when you purify them from the gel you still get those uncut plasmid, then when yo ligate and transform there should be more clones because there are thses plasmid supercoiled that need not to be ligated and just tranform as a usual plasmid. And also, I use those probably "mixed" plasmid to transform without ligating, not single grow which means there is not a significant amount of uncut DNA. So one great possibility is that when you purify fromn the gel, the incomplete washing get agarose gel component mixed into the plasmid which inhibit the ligation. So the more pure and efficient your cut the less possible to be ligated.
Maybe I'm wrong somewhere but by far I couldn't see better explains