Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Designing primers with recognition sites of restriction enzymes

Designing primers with recognition sites of restriction enzymes - PCR - Polymerase Chain Reaction Forum

Designing primers with recognition sites of restriction enzymes - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #11  
Old 01-01-2012, 02:04 PM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

SEQUENCE:
5'- CCG GGA TCC CGC GCG AGG ATG AGT TAT ACT GTC GGT AC -3'
COMPLEMENT:
5'- GTA CCG ACA GTA TAA CTC ATC CTC GCG CGG GAT CCC GG -3'
LENGTH: 38
GC CONTENT: 60.5 %
MELT TEMP: 69.3 C
MOLECULAR
WEIGHT: 11735.6 g/mole
EXTINCTION
COEFFICIENT: 363500 L/(molecm)
nmole/OD260: 2.75
g/OD260: 32.29

SEQUENCE:
5'- CCC GGT ACC CTA GAG GAG CTT GTT AAC AGG C -3'
COMPLEMENT:
5'- GCC TGT TAA CAA GCT CCT CTA GGG TAC CGG G -3'
LENGTH: 31
GC CONTENT: 58.1 %
MELT TEMP: 65.6 C
MOLECULAR
WEIGHT: 9521.2 g/mole
EXTINCTION
COEFFICIENT: 295700 L/(molecm)
nmole/OD260: 3.38
g/OD260: 32.20

Temperature wise it look ok, however still remember the self dimer and hetero dimer? It is not good. Very stable self/hetero dimer.

the problem is your forward primer 5' end. What about just synthesizing one primer?
Reply With Quote
  #12  
Old 01-01-2012, 05:06 PM
obama's Avatar
Graduate Student
Points: 2,830, Level: 34 Points: 2,830, Level: 34 Points: 2,830, Level: 34
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Hannover Germany
Posts: 145
Thanks: 2
Thanked 25 Times in 21 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

Try to do gradient PCR to optimize your pcr and try different primer concentration
Reply With Quote
  #13  
Old 01-02-2012, 04:19 AM
saranyah's Avatar
Volunteer
Points: 1,194, Level: 19 Points: 1,194, Level: 19 Points: 1,194, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 30
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

Thanks for such an elaborate calculation... I'll try re-synthesizing the forward. Should the annealing temperature or time be manipulated?
Reply With Quote
  #14  
Old 01-02-2012, 04:21 AM
saranyah's Avatar
Volunteer
Points: 1,194, Level: 19 Points: 1,194, Level: 19 Points: 1,194, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 30
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

What kind of changes should be done in the cycle? Should the time of each cycle be increased/decreased?
Reply With Quote
  #15  
Old 01-02-2012, 04:08 PM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

Quote:
Originally Posted by saranyah View Post
Thanks for such an elaborate calculation... I'll try re-synthesizing the forward. Should the annealing temperature or time be manipulated?
DON'T re-synthesize the same forwar primer, it have TOO stable forward 5' end homo/heterodimer. If you do have it optimized using your current forward primer do let us now.
Reply With Quote
  #16  
Old 01-03-2012, 09:16 AM
saranyah's Avatar
Volunteer
Points: 1,194, Level: 19 Points: 1,194, Level: 19 Points: 1,194, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 30
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

I cant understand what you have said sir? Should I synthesize another primer or try with the same?
Reply With Quote
  #17  
Old 01-04-2012, 02:51 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

you should design another forward primer with less G and C on the 5' end. Please do use the link i posted to check for homo/hetero dimer to avoid the same situation from happening again. Do you have any collegue in the same lab who have experience in primer design and can help?

or you could try your luck by optimizing your pcr primer as suggested by Obama if you cannot order another primer. Anybody in your lab with experience with pcr optimization?
Reply With Quote
  #18  
Old 01-04-2012, 03:49 AM
saranyah's Avatar
Volunteer
Points: 1,194, Level: 19 Points: 1,194, Level: 19 Points: 1,194, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 30
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

Unfortunately I'm in a bioinformatics lab and my seniors are not exposed to molecular biology work... I'll try optimizing the PCR and also if I'm having fund I'll synthesize a new forward... Thanks for offering ideas....
Reply With Quote
  #19  
Old 01-04-2012, 04:28 AM
butters's Avatar
M.D/Ph.D
Points: 4,938, Level: 47 Points: 4,938, Level: 47 Points: 4,938, Level: 47
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2008
Location: Malaysia
Posts: 315
Thanks: 25
Thanked 68 Times in 63 Posts
Default Re: Designing primers with recognition sites of restriction enzymes

do update us on the progress, especially me as I am really curious what possible experimental parameter can actually remove that kind of strong binding.
Reply With Quote
Reply

Tags
designing , enzymes , primers , recognition , restriction , sites


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Help with restriction enzymes!! grandhotel Biochemistry Forum 0 03-26-2011 12:23 PM
Help with Restriction enzymes!! grandhotel DNA Forum 0 03-26-2011 12:21 PM
Original Biotech Rap Restriction Enzymes Funny Video dnamolecule Science and Lab Jokes 0 11-29-2007 04:39 AM
Restriction sites. Ezhil Subbian Protocols and Methods Forum 1 06-29-2006 02:43 AM
Introduction of restriction sites via extended primers? Peter Frank Protocols and Methods Forum 6 08-14-2004 06:34 AM


All times are GMT. The time now is 09:42 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16659 seconds with 16 queries