PCR problems - (Dec/10/2003 )

Hi everybody!

My problem is I'm unable to amplify my DNA by PCR. I explain... I have a DNA with an insert. By digestion with specific enzyme, I know that my insert is there. But when I use a primers specifics at my vector to check this insert, I obtain only 1 band like if I dont have the insert.
My PCR conditions are good because I test with an other DNA construction and the insert is amplified. You can help me...now I dont know what cant I do!

Thanks

Hi,

How big is your insert? You said you got a single band which is the size without insert. I guess the size of your band is only dozens.

If your insert is big, did you use polymerase and cycling condition for long-distance PCR?

SAGE

hello, PCR-aldy:

In your experiment, there may be DNA contamitant ! In our last DNA construction experiment , We constructed one protein overexpression vector ,the insert DNA is about 700 bp. After we constructed , the insert of vector was asertained by PCR and restriction digestion .Nevertheless, the insert DNA sequen is not our interest by the DNA sequencing

Hi Tanghuoxian,

I think that you already have this problem no? Now how you proceed to check if your construction is good (if your insert is in your vector) if is impossible to check by PCR or by digestion with specific enzymes?

I know that my first insert is there by digestion but not by PCR. But for my second insert, how is possible to check? I have many problems with my ligation and I need to check each colony after transformation with competents cells to know which one is good and by pcr this is fast!

You have a suggestion for me?

Thanks