2 replies to this topic

[zzz2]

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

Edited by zzz2, 18 October 2012 - 01:29 PM.

[bob1]

Add the RE sites to the 5' end of the primer - so your part 3 is correct.  You usually also need to include a few bases (3-6) to the 5' of the RE site too, so as to allow the RE something to bind to.  The gene specific part of the primers would usually need to be longer than that to get a specific product.

There is no need for programs to generate these primers- you have very tightly defined locations at the beginning and end of your GOI.

[v.sabastian]

zzz2, on 18 October 2012 - 01:27 PM, said:

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

Hi,

Even I would suggest you to attach a restriction enzyme to your designed primers and then amplify the plasmid and ligate it. So, the answer to your third question is 'Yes'. Also, make sure that the enzyme should be added at the 5' end of the primers.

The primers should be between 25 and 45 bases in length, with a melting temperature of ≥78°C. The desired mutation should be in the middle of the primer with ~10–15 bases of correct sequence flanking both the sides. And both the mutagenic primers should anneal to the same sequence on opposite strands of the plasmid.

Here is a freeware to design specific mutagenic primers:
http://www.premierbi...esis/index.html

Sabastian

Edited by v.sabastian, 10 December 2012 - 04:55 AM.