Help : no positive colonies - (Jun/12/2006 )
I added 3 extra bp in front of the restriction sites in the PCR product.
I don't thing the problem comes from the cels because another person obtained good results with the same cells and I have tried with electro and chimical competent cells.
I have already changed my culture media.
Other ideas?
I did not understand clearly, you are getting the two bands when you screen the clones ? or is it beforehand .please clarify
if you are getting the two bands while digesting the plasmid vector alone, check the plasmid, it may be wrong.
next, if you are getting the bands only during clone screening and not in the plasmid vector alone, change the enzyme and the buffer, try a new company.
both Eco RI and Bam HI have star activity therefore long incubations can give erroneous results. Check NEB catalog for more info.I also did get some erroneous result because the buffer was wrong. though each might work in the buffer but, pH of the buffer may influence star activity.
I did not understand clearly, you are getting the two bands when you screen the clones ? or is it beforehand .please clarify
if you are getting the two bands while digesting the plasmid vector alone, check the plasmid, it may be wrong.
I have the two bands after digestion of the plasmid.
How checking the plasmid?
Can you give me a protocol for CIP treatment please?
NEB Calf Intestinal Phosphatase - just check the company website....although many other people on this forum recommend shrimp alkaline phosphatase instead
also - if 2 bands after a double digest - implies that something is being cut out of the vector - maybe check your size after one cut to be sure.....
oh, and by checking we mean either:
1. sequencing
2. size check of linear plasmid (ie. cut with one enzyme only)
After one cut : two bands...as after double digestion...
If you see two bands very close together and you assume one of the bands is a fragment cut out of the vector then your original plasmid would be huge (sth. like 19 kb).
You can check your vector by cutting it with different combinations of enzymes and see if you get the expected fragment lengths, e.g. one enzyme in the MCS and one elsewhere in the vector. Check if you get additional bands here as well.
Treatment with CIP is simple. Anyways, if you have a choice, go for NEB Antarctic Phosphatase. I would purify the vector first to get rid of restriction enzyme and buffer. Then take x ul vector (up to 17 ul if concentration is low) + 2 ul 10x Antarctic Phosphatase Buffer + 1 ul Antarctic Phosphatase (= 5 Units). Add H2O to a volume of 20 ul. Incubate at 37 °C for appr. 25 - 30 min. Heat-inactivate the Antarctic Phosphatase at 65 °C for 5 min. Purify vector again. Ready for ligation.
AP works also in NEB buffers 1-4, EcoRI and BamHI if you add 10 x AP buffer, so theoretically you don`t need to purify your vector before you treat it with CIP/AP, but for me the treatment was less effective then.
I have cut my vector with other enzymes than BamHI and EcoRI and I get the expected fragment lengths
AP works also in NEB buffers 1-4, EcoRI and BamHI if you add 10 x AP buffer, so theoretically you don`t need to purify your vector before you treat it with CIP/AP, but for me the treatment was less effective then.
I think I will try to dephosphorylate my plasmid.
At our lab, we have a phosphatase alkaline from calf intestine (boehringer).
For inactivation, they say :
"add 1/10 volume of 200mM EGTA to the reaction assay and heat to 65°C for 10 min. To achieve complete inactivation of phosphatase, an extraction with phenol/chloroform/isoamylalcohol should be performed."
You don't add EGDA? Is it specific to my phosphatase?
To purify the vector, do you perform a phenol/chloroform/isoamylalcohol extraction or do you purify on a column?
For ligation, do you thing 100 ng plasmid in 20µl is enought? After : transformation with 5µl in 100 µl chimical competent cells? is it all right?
Thank you for your answers and advices.
I did not understand clearly, you are getting the two bands when you screen the clones ? or is it beforehand .please clarify
if you are getting the two bands while digesting the plasmid vector alone, check the plasmid, it may be wrong.
I have the two bands after digestion of the plasmid.
How checking the plasmid?
hi ,
I agree with "aussieuk" .Sequence it or digest with single enzyme or with two enzyme and see whether the insert that is released corresponds to the size given the vector map of the plasmid. suppose there is one site of Eco RI at 1kb and one site of Sal I at 2.5Kb ,and these are single cutters, then doing a double digestion you will get a insert release of 1.5kb and rest will be the backbone, you can add up the two and see if it comes to the reqd. size of the plasmid. choose single cutters only as otherwise you will get a complex pattern.
If you are using some commercial plasmid , you can alawys order a new vial and restart. believe me, it is better starting again then wasting time trying to find out what happend.
good luck
Antarctic phosphatase can be heat inactivated. NEB says that CIP can`t be heat inactivated completely. So maybe thats why you have to add EGTA.
I usually purify over the column, but please follow the protocol given for your CIP. They will have reasons to advise you to purify it with Phe/Chl/Isoa.
100 ng plasmid is enough for ligation. Transform 5-10 ul.