Difficulties in transformation - (Jan/12/2001 )
I'm trying, since 6 months or so, to transform my bacterial cells (commercially available: i.e DH5alpha, DH10B, XL1-Blue and/or SURE). I've check the ligation reaction (3 times more moles of insert than vector for a least 500ng total DNA!!!) and everything is OK: we can see an up-shift. Unfortunetly I get nothing or some contamination: i.e. a bacterial specie that is (depending on the strain used) ApR CmR KanR StrepR and/or TetR !!! this monster should be a Pseudomonas sp. The media used or always freshly made and at correct antibio. final concentration. The cells used are viable and competent (I usually get a lawn after a transfo with less thant 0,5ug of the vector). This is it, so if there's someone on the globe that can give me some advise you're desesperately WELCOME.
It's most likely to be your ligation reaction and not the transformation that is the problem. You say you use at least 500 ng DNA, for what volume? It is probably way too much! You should have <10ng per ul. It is a common mistake for people to use too much DNA for ligation - too much DNA gives linear ligation product that is difficult to transform instead of forming circularised DNA. Also do not use TBE for your gel when you are doing gel purification of digested fragments. You also did not properly check for the transformation efficiency.You should use, say 1 ng of uncut plasmid, transform and then so serial dilution of your cells, plate andcalculate the transformation efficiency.
Maybe you should try a new vector/antibiotic combination. I am in no way connected with Novagen, but they have a great ligation/transformation kit called Perfectly blunt. The kit uses a vector called pstblue-1, which is an amp resistant plasmid. (But try to use carb. in the media if you can)
Agreed, I made the mistake of using too much insert DNA in a blunt ended ligation reaction and a lot of the plasmids had more than one target sequence ligated to one another within them. Try using a 100 fragments for every 1 vector ratio. This takes a little calculating to figure out # of fragments from the mass of the sample, but it can be done; and it's a good place to start. good luck.
Borate in TBE inhibits ligation reaction if it is not removed properly. Took me some time to find out, and when I switch to TAE, the increase in the number of transformants is amazing. I guess you must have been removing borate more efficiently than I have.
not really related but why not use TBE when excising DNA fragments from gels, I've been doing this and it has worked fine so far???
I read all the replies about my message on who to improved my transfo.I still one or two controls to do, and the answer could simply be that the WATERI been using for so long is simply not correct for transfo!!!!!I used milipore (pr mQ) instead of distilled water!!! I read in this forum a message of someone that I have the same prob. than me except that he or she performed electropo.but the cells I been washed with mQ water!!! So be advised folks never trust the water you use!!! Ha Ha Ha ...
I may be wrong but is millipore water not more 'pure' then distilled as it has ben filtered as well as distilled! Did you autoclave to water you used originally?
Milipore water is suppose to be a distilled water which had been process throught an serie of ions exchange cartridges. So it mightbe Ğultra pureğ but one of my collegue had obeserved that using thatkind of water render less fluid the bacterial membrane!!! So performingtransformation which have a lot to do with fluidity, it maybe notsuitable to use milipore water. I personnaly believe that a double distilled water would better. But still, I did not solve my f...problem, time fly and I will be force to surrender. I do not that muchtime left (I supposed to make my deposit in september) LIFE'S A BITCH
Buy competent cells. I don't normally recommend buying but it's OK if you have not time to find out what the problem is.