
issues when designing PCR primers using PRIMER3
#1
Posted 16 October 2012 - 02:10 PM
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
Posted 16 October 2012 - 02:35 PM
#3
Posted 16 October 2012 - 02:45 PM
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
Posted 16 October 2012 - 02:51 PM
#5
Posted 19 October 2012 - 09:36 AM
K.
Edited by Neanderthal, 19 October 2012 - 09:43 AM.
#6
Posted 20 October 2012 - 01:08 PM
(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
Posted 29 October 2012 - 02:55 AM
#8
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
Posted 10 December 2012 - 05:51 AM
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