Ligation and transformation problem - (May/13/2011 )

Dear all,

I need your suggestions.
For gene expression needed, i cloned my gene that contain restriction site (NcoI and XhoI)in the terminal to pET28a (+) vector. Before i ligate my gene target into vector, i digested both my target gene and pET28a (+) vector with Restriction Enzyme NcoI and XhoI at 37oC about 4 hours. After that, i inactivated the enzyme by heating them at 65oC about 20 minutes and purified using spin column from QIAgen.

Purified pET28a (+) and my target gen and then ligated by T4DNA ligase at 4oC overnight. To check the ligation process, i conducted PCR. I used primer:

1. T7 promoter primer and T7 terminator primer (primer of vector)
2. T7 promoter primer and my reverse primer (primer sequence at 3'terminal of my gene target)
3. My forward primer (primer sequence at 5'terminal of my gene target) and T7 terminal primer

and i got the result:
1. Using T7 promoter and T7 terminator primer, i can not get DNA band (negative result)
2. Using T7 promoter primer and my reverse primer, i got DNA band (very thin band)
3. Using my forward primer and T7 terminal primer, i got DNA band (bold band).

Can you explain to me, ligation process completely done or not? because using T7 primer, there was no DNA band.

For my point of view, the ligation process worked properly. So i transformed the ligation product (2 micro) to E.coli TOP10. I got the colonies, but detection using my forward primer and T7 terminal primer, i got negative result (no band).

Can you also explain, what the problem with my procedure?

Thanks for your nice suggestion and problem solving.

hadi

What kind of PCR do you use, colony PCR or plasmid PCR? And the size of band is what you expect? Sometimes colony can't work very well. And why don't you use both of your primer for PCR?

For your ligation, do you have ligation control (vector without insert)? Use this control to do transformation at that same time with your regular ligation (vector plus insert). If the plate of control is empty, while the plate of vector plus insert get some colony, your ligation is sucessful. The you can pick some colony to test. Normally, I use restriction enzyme to digest first, then sequence it.

good luck!

George


quote name='snhadi' timestamp='1305272401' post='109665'>
Dear all,

I need your suggestions.
For gene expression needed, i cloned my gene that contain restriction site (NcoI and XhoI)in the terminal to pET28a (+) vector. Before i ligate my gene target into vector, i digested both my target gene and pET28a (+) vector with Restriction Enzyme NcoI and XhoI at 37oC about 4 hours. After that, i inactivated the enzyme by heating them at 65oC about 20 minutes and purified using spin column from QIAgen.

Purified pET28a (+) and my target gen and then ligated by T4DNA ligase at 4oC overnight. To check the ligation process, i conducted PCR. I used primer:

1. T7 promoter primer and T7 terminator primer (primer of vector)
2. T7 promoter primer and my reverse primer (primer sequence at 3'terminal of my gene target)
3. My forward primer (primer sequence at 5'terminal of my gene target) and T7 terminal primer

and i got the result:
1. Using T7 promoter and T7 terminator primer, i can not get DNA band (negative result)
2. Using T7 promoter primer and my reverse primer, i got DNA band (very thin band)
3. Using my forward primer and T7 terminal primer, i got DNA band (bold band).

Can you explain to me, ligation process completely done or not? because using T7 primer, there was no DNA band.

For my point of view, the ligation process worked properly. So i transformed the ligation product (2 micro) to E.coli TOP10. I got the colonies, but detection using my forward primer and T7 terminal primer, i got negative result (no band).

Can you also explain, what the problem with my procedure?

Thanks for your nice suggestion and problem solving.

hadi