Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

issues when designing PCR primers using PRIMER3


  • Please log in to reply
8 replies to this topic

#1 weihua

weihua

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 16 October 2012 - 02:10 PM

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.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,738 posts
400
Excellent

Posted 16 October 2012 - 02:35 PM

Primer3 should give you the sequence as you would order it (i.e. already reverse complemented). If you look at where the program shows the primers to be according to the sequence you submitted, the reverse primer should show the reverse complement of the sequence.

#3 weihua

weihua

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 16 October 2012 - 02:45 PM

Thank you. I use NCBI primerBLAST. It uses primer3.
I assume the output of the reverse primer need not to be changed, right?
the output look like this:
sequence 5'-3'
Forward primer ACTGACTG
Reverse primer CTGACTGA
So I should directly order as above? right?

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,738 posts
400
Excellent

Posted 16 October 2012 - 02:51 PM

Yep.

#5 Neanderthal

Neanderthal

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 42 posts
1
Neutral

Posted 19 October 2012 - 09:36 AM

Yes, bob1 is right, Both Primer3 and NCBI-Primer BLAST gives hte primers in 5'-3' direction. As far as I know Primer Express also gives it in 5'-3' format.

K.

Edited by Neanderthal, 19 October 2012 - 09:43 AM.


#6 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 20 October 2012 - 01:08 PM

Also easy way to check this is to try and find the primers in input sequence. You should find there forward primer, but not the sequence of reverse. If you use some online reverse-complement tools on your reverse primer, you should now be able to find it in the sequence.
(but note it's just common agreement to call the first primer on the sequence forward and second reverse, and all common primer designing tools do it this way, but in some special circumstances reversed orientation of the first primer on sequence could be choosen and also corresponding with that, the second primer orientation would be identical to input sequence, but in that case it would be appropriate to call the first primer reverse and the second forward, but you can always blast the primers and check whether they are on the same sequence in opposite directions [important is to know that the source sequence in Blast can be sometimes in reverse orientation])

And last note, primer 3 displays the position of both primers ranked 1 on a input sequence, you it's most easier to check it there.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#7 weihua

weihua

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 29 October 2012 - 02:55 AM

Thanks for all.

#8 v.sabastian

v.sabastian

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 10 December 2012 - 05:01 AM

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

Edited by v.sabastian, 10 December 2012 - 05:14 AM.


#9 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 10 December 2012 - 05:51 AM

No it's not possible that the primers may not have been designed accurately. Are you trying to spam inconspicuously for NetPrimer? Your post have no sense.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.