Too many colonies - (Jan/23/2006 )
hi
when i did a transformation i got a plate which looked like a control plate. i have screened 30 colonies and have seen only native plasmids so far
i would like to know
1. the probability of getting a recombinant
2. any other method of screening recombinants... iam using alkaline lysis
any help will be appreciated
p.s : the vector i am using does not have lac Z .. so blue white screening is out of question
How are you cloning? Is it sticky end or blunt end cloning? Is it sticky, is it one enzyme or two enzymes you are using to digest the vector? Also, I presume your insert has no selectable marker like an antibiotic cassette? With no selectable marker, you may have to screen heaps of colonies due to vector religation. I a not sure what the ratio of background to positive clones would be, I guess it varies. You can certainly reduce background by dephosphorylating the vector or using two enzymes to digest the vector resulting in incompatible ends. Therefore, theoretically...only plasmids with inserts can be ligated.
One method would be two screen colonies by PCR in lots of 12. I use microtitre blocks that hold 1.5 ml of media and then inoculate a colony per well using sterile toothpicks. I let the cultures grown and then screen row by row (12 wells per row), by taking 50 ul of culture from each well and pooling the cultures. I then spin, resuspend the cells in 50 ul of water and use 2 ul in a 20 ul PCR. Any rows that are positive, I then go back and screen by individual well.
I also wonder how you prepared the plasmid for transformation. After your initial digest, did you purify the vector? How long did the digest run and are you sure that the combination of enzymes you used work well in the buffer you used? If your plate looks like a control plate it is most likely that you have had improper digestion and are doing transformations with the original plasmid intact. So my questions are:
1) Which enzymes did you use and in which restriction buffer?
2) For how much time did you allow the digest to incubate?
3) Did you run your digest on a gel to ensure it is indeed digested and then purify the vector band?
I would suggest forgetting about screening your lawn of cells and go back to the digest part. It will probably save you a lot of time and supplies.
One method would be two screen colonies by PCR in lots of 12. I use microtitre blocks that hold 1.5 ml of media and then inoculate a colony per well using sterile toothpicks. I let the cultures grown and then screen row by row (12 wells per row), by taking 50 ul of culture from each well and pooling the cultures. I then spin, resuspend the cells in 50 ul of water and use 2 ul in a 20 ul PCR. Any rows that are positive, I then go back and screen by individual well.
thanx ML1975 and captain DNA for your reply...
I prepared the plasmid using a binding column which gives a purified plasmid. then i did a double digestion of plasmid + insert with BamH1 and HindIII by keeping it at 37 c for 3 hrs. i used a hind III buffer which has worked previously. i did not check the digest on a gel but proceeded directly for quick ligation and transformation.
for how long can a digestion mix be stored?
I prepared the plasmid using a binding column which gives a purified plasmid. then i did a double digestion of plasmid + insert with BamH1 and HindIII by keeping it at 37 c for 3 hrs. i used a hind III buffer which has worked previously. i did not check the digest on a gel but proceeded directly for quick ligation and transformation.
for how long can a digestion mix be stored?
I think your problem in this case is the lack or purification of the digested plasmid. Do a 1-3 hour digest (I have done overnight before and as long as over-weekend and had no problems) and run the entire digest on a gel (maybe include two single digest (Bam and Hind) as controls). From there isolate your digested vector band from the double digest lane and extract the DNA from the gel using whichever method you so desire. Use that purified DNA for your ligation. I don't know how you are preparing your insert but if it is in another vector, do the same digest and gel purification. Also look on your single digest controls to make sure that both enzymes are indeed working.
I personally have never gone straight from digest to ligation without a purification step but that is just how I do things. Perhaps this will help you. Best of luck
-Cap
thanx ML1975 and captain DNA for your reply...
I prepared the plasmid using a binding column which gives a purified plasmid. then i did a double digestion of plasmid + insert with BamH1 and HindIII by keeping it at 37 c for 3 hrs. i used a hind III buffer which has worked previously. i did not check the digest on a gel but proceeded directly for quick ligation and transformation.
for how long can a digestion mix be stored?
I think your problem in this case is the lack or purification of the digested plasmid. Do a 1-3 hour digest (I have done overnight before and as long as over-weekend and had no problems) and run the entire digest on a gel (maybe include two single digest (Bam and Hind) as controls). From there isolate your digested vector band from the double digest lane and extract the DNA from the gel using whichever method you so desire. Use that purified DNA for your ligation. I don't know how you are preparing your insert but if it is in another vector, do the same digest and gel purification. Also look on your single digest controls to make sure that both enzymes are indeed working.
I personally have never gone straight from digest to ligation without a purification step but that is just how I do things. Perhaps this will help you. Best of luck
-Cap
hey captain DNA thanx a lot.. am pretty new to this forum and did not expect a reply pronto!
p.s i did purify my plasmid after diestion but probably i have lost the digested plasmid... i should check that out...
Another approach we are using to reduce/eliminate background for cloning is to avoid preparing the plasmid backbone by cutting a whole plasmid. Instead, prepare the backbone by PCR, using primers which prime to the cloning site and contain the restriction sites you plan on using. Include 6-10 bp of 5' sequence to allow the PCR product to be cut at those sites.
PCR very highly diluted plasmid as a template with these primers and check length on a gel. Purify the DNA (precipitation, column, whatever). Now, you can simply mix the plasmid and insert DNA, cut with both enzymes, ligate, and go. The backbone will not ligate with itself. There is essentially zero uncut plasmid (only the remnant of the PCR template, at very very low concentration). There is no chance of trashing the DNA by gel visualization or phosphatase treatment.
Interesting! That sounds like a pretty cool approach. Does it usually work well for you?
PCR very highly diluted plasmid as a template with these primers and check length on a gel. Purify the DNA (precipitation, column, whatever). Now, you can simply mix the plasmid and insert DNA, cut with both enzymes, ligate, and go. The backbone will not ligate with itself. There is essentially zero uncut plasmid (only the remnant of the PCR template, at very very low concentration). There is no chance of trashing the DNA by gel visualization or phosphatase treatment.
i am trying to clone my gene into an expression vector. can i prepare the backbone by PCR ?
what is the specificity of creating the bacbone this way?
do i require a high fidelity polymerase?
do i have to sequence and check after PCR?