Hallo,

I understand a normal really simple PCR reaction. But now i have a small problem.

My gene:

5' (Rest of genome)TTATCCT........TACTCAT(Rest of genome) 3'

In my vector there are the following restriction sites:

HindIII

5'-A|AGCT T-3'
3'-T TCGA|A-5'

XbaI

5'-T|CTAG A-3'
3'-A GATC|T-5'

The promoter is before the HindIII restriction site.
Now i have to create primers to add sticky ends to my GOI to integrate the reverse complement in the right direction.

1. How do you do this? With programs?
2. It doesn't matter if there are hair pins or anything else. My intention is to understand it.
3. Are these primers correct?

5‘ TCTAGA TTATCCT 3‘
5‘AAGCTT ATGAGTA 3‘

1. part = restriction site for sticky ends

i am getting a low PCR product ! I have tried reducing the annealing temp, increasing the primer and DNA template conc. too. But still quite low. Anything I should try ?

When I was designing primers using PRIMER3
The screen outputs like follows
Forward primer ACTGACTG
Reverse primer CTGACTGA

I was wondering do I need to reverse and complement my reverse primer when ordering my primers?

For example, I should order TCAGTCAG for my real reverse primer
Thank you.