troubles in DNA digestion - (Feb/03/2012 )
Hi all,
I've been trying to digest pGEX-4T-1(4kb) and DNA sequence of Mycoplasma (514pb) using SalI and EcoRI for 4 weeks.. But i can find nothing after runnig an agarose gel (1%).
I know that 1U of the enzyme digest completly 1µg of DNA in one hour at 37°C. but I'm not able to exploit this knowledge
I'm using the following protocol;
H2O 5µl
OPA buffer 2x 1µl
DNA / pGEX 4µl
SalI 15U/µl 0.01µl
EcoRI15U/µl 0.01µl
I tryed diffrent times of incubation at 37°C: 60; 45 and 30 minutes.
I changed diifrent volumes of DNA.
but i still find nothing on agarose gel
questions are:
1- How can i know if my enzymes are ok (as they ar not new)?
2- as i can't know the concentration of my plasmide neither my DNA, how can i calculate the number of unit of enzyme i'm supposed to use?
( in the pic of agarose gel, i put 3 µl of pGEX as positif control : non digested plasmid)
If any one can help, I'll be really grateful
Its difficult to pipette 0.01ul of enzyme.
You need to have a positive control for the enzymes to know if they digest it properly.
there are ways to calculate DNA from agarose , by running different volumes of known conc. of ladder and comparing the intensity of your sample to the ladder.
I would suggest digesting much more DNA and digest it in 20-50ul
No one can pipet .01 ul of enzyme. I'd suggest that you failed to add any. As scolix suggests, do a digestion in 50 ul. I don't know what OPA buffer is, but if it is really a 2x buffer, then you added it at 10x dilution. More likely it is a 10x buffer and you wrote it incorrectly. I would add 1 ul of each enzyme.
.
scolix on Fri Feb 3 21:14:24 2012 said:
Its difficult to pipette 0.01ul of enzyme.
You need to have a positive control for the enzymes to know if they digest it properly.
there are ways to calculate DNA from agarose , by running different volumes of known conc. of ladder and comparing the intensity of your sample to the ladder.
I would suggest digesting much more DNA and digest it in 20-50ul


You are confused. You should be using much more enzyme. Do the reaction this way:
39 ul water
4 ul of your DNA
5 ul of OPA buffer (it really is 10x, unless you have diluted it)
1 ul of SalI
1 ul of EcoRI
Mix, digest for 1 hour at 37 degrees. Yes, there is an excess of enzyme. Yes, this is important and intentional. Yes, you could do this in a lower volume, but no, that would not be a good idea.
phage434 on Sat Feb 4 16:40:53 2012 said:
You are confused. You should be using much more enzyme. Do the reaction this way:
39 ul water
4 ul of your DNA
5 ul of OPA buffer (it really is 10x, unless you have diluted it)
1 ul of SalI
1 ul of EcoRI
Mix, digest for 1 hour at 37 degrees. Yes, there is an excess of enzyme. Yes, this is important and intentional. Yes, you could do this in a lower volume, but no, that would not be a good idea.



You should transform into a cloning strain such as Top10 or DH5a rather than BL21. Then, prepare plasmid from the cloning strain and then transform into BL21.
You should only need about 20 ng of digested DNA in your ligation mix.