Changing the vector - (Aug/05/2014 )

So, I'm still not clear. Did you are did you not see a correct 4 kb band after your PCR?

Yes I did phage 434. One more point guys The primers I am using has 5' phosphate. Do you think this contribute anyway to the low yield?

I can't see why it would but that doesn't mean anything :-P

Now what is the problem this time? I have repeated The PCR step by lowering the annealing temp to 62 degree. 

I can not see any band.

The there cycling condition 

98 30sec

98  10sec

                    35cyc

62   2min

 

72 10min

RNA woman on Wed Oct 1 05:52:59 2014 said:

Now what is the problem this time? I have repeated The PCR step by lowering the annealing temp to 62 degree. 
I can not see any band.
The there cycling condition 
98 30sec
98  10sec
                    35cyc
62   2min
 
72 10min

I'm confused by your cycles here. It may be showing up weird because I'm on my phone but it should look something like this:
98C 30 seconds (initial denaturing)
Followed by 35 cycles of:
98C 10 seconds (denaturing)
62C 10 seconds (annealing)
72C 2 minutes (extension)

Then after those cycles are all done
72C 10 minutes (final extension)

Again though, if you are already getting a visible band using your other conditions, simply doing a TOPO cloning may be the way to go.

For the Topo cloning. It is written as note to not use 5 phosphates primers for PCR that PCR product synthesised will not ligate. I don't understand why? I will remove the primers with the column and the PCR product it self will not have 5' phosphates. 

Your PCR product will have 5' phosphates if the primers have those phosphates. I was unaware of that issue with TA cloning, since I never use it. You can remove the 5' phosphates with shrimp alkaline phosphatase or antarctic phosphatase. Likely you can directly add 1-2 ul of SAP to your PCR reaction following PCR, without purification. AAP will require adding its buffer, which contains zinc.

I never knew that either. I was just looking it up. You learn something new every day!

Bio-Lab,

I found something here. The primers that you suggested and I used to amplify the gene. have EcorV and NdeI not NheI restriction site.

I found also EGFR has two NdeI sites in the meddle of its sequence. Could you confirm please?