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There have been 3 items by v.sabastian (Search limited from 15-November 18)


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#146497 issues when designing PCR primers using PRIMER3

Posted by v.sabastian on 10 December 2012 - 05:01 AM in PCR, RT-PCR and Real-Time PCR

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.


Hi,

Yes, it is possible that the primers may not have been designed accurately. I would suggest you to evaluate your primers using NetPrimer, which is freely accessible.

Here is the link to NetPrimer:

(http://www.premierbi...rimer/index.htm)

Also, I found a great resource that claims to be exclusively designed for bacterial identification:

(http://www.premierbi...ation/realtime-PCR/species-identification.html)
Hope this helps.

Sabastian



#146496 Primer for a gene to create sticky ends and ligate an gene into a vector

Posted by v.sabastian on 10 December 2012 - 04:54 AM in PCR, RT-PCR and Real-Time PCR

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



#146495 Low PCR product

Posted by v.sabastian on 10 December 2012 - 04:49 AM in PCR, RT-PCR and Real-Time PCR

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 ?


Hi,
There can be many contributing factors for getting a low PCR product. You can troubleshoot the following:
1) Check whether the primers you designed are perfect complementary to the DNA strands. Perhaps they are not specific which is one of the major reasons for low PCR product yield. Moreover they should not be self complementary or each others complement. If you designed them manually, I would suggest using a good primer design software. There are many tools available in the market. I personally prefer and use Primer Premier to design my primers and the tool has not disappointed me so far. Here is its link which I found on their website: http://www.premierbi...sign/index.html
2) You can try to reduce the volume of sample in the reaction mix if required and carry out another ethanol precipitation with the samples.
3) Try to optimize Mg2+ ion concentration and use a good proofreading enzyme with high fidelity.
All the best for your assays.


Cheers!!
Sabastian




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