A Digestion problem - (Nov/17/2004 )

I have a problem with digestion.
What I did are: first I did pcr to amplify my insert and run a small amount of the insert on the gel. the insert showed the right size. Then I purified my insert and modified the end by BamH I. After that I run on the gel again. But I found two bands : one is what I want and the other is a bigger size faint band. How could this happen?

Any one has ideas about this and what should I do next?

Thanksa lot!

Hi
"you have observed two bands of which one is of your interest
and the other of big size."

have u run your undigested template along with digestion product, to clarify whether any undigested template is left out in restriction digestion process. if so, please prolong the incubation time for complete digestion

good luck
ravi, INDIA

Thank you for your reply!
The problem is there are only several bp difference between the digested one and undigested one. So it is hardly to tell them.
I incubate for 2hs. I am wondering is there any way to enhance the diggestion efficiency?

I have annother question is about ligation.
According to the instruction , the vector and the insert ratio should be1:3. However, some people said they never measured the concerntrations of vector and insert , so they just mix them by their experience. Which one I should follow?

Thanks again!

It may be better to elute DNA with water, so that enzymes have the right buffer concentration - some enzymes are very sensitive to the conditions...

I think, it is always better to calculate the right ratio. I would always measure DNA-concentrations for ligation!

Yes it is !
it s necessary to check the conc. of insert and vector, and what you heard is right to take in 1:3 ratio (vestor : Insert). Pls. refer Molecular cloning by Maniatis, 1982 for detailed protocols.

Yes, as mentioned earlier by Janina, while purification try to elute in water. Coz... its assumed that EDTA some time interfere with digestion process.

Good luck

ravi
INDIA

Thank you very much you all of your reply!
I will absolutely follow you advice.
Another simple question is how many ng vector or insert you usually use?you measure the con. on machine or estimate on run gel? you know someone told me that if the con. is too low(ng) , the machine in my lab can not give the accurate measurement.

Thanks!

This is right, the extinction of DNA (260 nm) should be between 0.1 and 0.01 - otherwise the result won't be precise enough.

For ligation, I use fmol (short calculation from ng to fmol...). Normally 5 fmol vector, 15 fmol insert. It is always the best to use as less ng as possible because the competent cells don't like so many DNA and the efficiency might be less...