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.


Single primer giving spurious band at the desired position after 3' RACE ..

Single primer giving spurious band at the desired position after 3' RACE .. - PCR - Polymerase Chain Reaction Forum

Single primer giving spurious band at the desired position after 3' RACE .. - 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
  #1  
Old 09-07-2009, 06:55 AM
ari ari is offline
Pipette Filler
Points: 75, Level: 1 Points: 75, Level: 1 Points: 75, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 6
Thanks: 1
Thanked 0 Times in 0 Posts
Post Single primer giving spurious band at the desired position after 3' RACE ..



Hiiii everybody....

I am a new member to this community and this oneís my first post.. actually i'm facing some problem in doing 3' RACE.. though i've designed the Forward Primers maintaining stringent parameters and have designed from the ROH of other organisms from the same group... when i performed the 3' RACE using the cDNA (synthesized using cDNA synthesis kit from Fermentas, in case of 3í RACE I didnít use any kit) in some cases i found band at the desired region along with various other multiple bands at different positions.. But to crosscheck it when i performed Negative Control PCR using just the Forward Primer in one reaction and Reverse Primer i.e. the Anchor primer in another reaction (using double conc. of the primer in each case) resp., to my surprise i got the band at the same desired region in one of the two reactions which i got when i did 3' RACE... tried with different primers but facing the same problem... i am really confused and dont know how to solve this kind of problem when the single primer is giving away the band at the desired position... Can anybody please suggest how to overcome such problem and a way to distinguish the desired band from those spurious bands at the same region... ???.........

Last edited by ari; 09-08-2009 at 07:59 AM.
Reply With Quote
  #2  
Old 09-08-2009, 08:00 AM
ari ari is offline
Pipette Filler
Points: 75, Level: 1 Points: 75, Level: 1 Points: 75, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 6
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

???????????????????????????????????........
Reply With Quote
  #3  
Old 09-08-2009, 03:03 PM
Graduate Student
Points: 3,129, Level: 36 Points: 3,129, Level: 36 Points: 3,129, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2008
Posts: 108
Thanks: 4
Thanked 48 Times in 42 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

I'm curious as to the method you've chosen for RACE. There was another poster here as well that used various components from Fermentas to do RACE manually, without a kit? Is this a new trend in RACE methods?

As far as I know, only a few companies make RACE kits as it's quite a delicate process. I've checked Fermentas' website, and I cannot find anything on RACE kit or components there. I would appreciate if you can clearly specify what exactly are the components from Fermentas that you are using to do your work.

As for similar bands appearing in both reactions with a single primer, perhaps it means that your PCR reagents/ machine/ pipette could be contaminated. Try starting your work from fresh.
Reply With Quote
The Following User Says Thank You to jonoave For This Useful Post:
ari (09-10-2009)
  #4  
Old 09-10-2009, 09:54 AM
ari ari is offline
Pipette Filler
Points: 75, Level: 1 Points: 75, Level: 1 Points: 75, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 6
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

Hiiiiiiiiiiii Jonoave.....

Thanxxx for replying....

...let me explain briefly how I am performing 3' RACE...
First of all i'm not using any RACE kit from Fermentas... i'm just using their cDNA synthesis kit comprising M-MuLV Reverse Transcriptase, RiboLock™ RNase Inhibitor, 5X Reaction Buffer, dNTP Mix (10 mM each), Oligo(dT)18 Primer, DEPC treated water, etc. along with Control RNA and Control primers just to synthesize the cDNA from the total RNA extracted. Now, previously i used to synthesize cDNA using Oligo dT 27 Primer but last time i synthesized using Oligo dT anchor primer. Using the resultant cDNA as the template i used to perfom the 3' RACE using the Gene Specific Primer (GSP) as the forward primer and the anchor primer as the reverse primer. These GSPs have been designed from the conserved region of the same gene from the other organisms of same class maintaining stringent parameters. Now when i performed PCR using these GSPs and anchor primer, in three cases i got the band at the desired region .. But as i reported before, when i crosschecked using only the GSP with the template in one set and using only the anchor primer with template in another set it (GSP) gave band at the desired region which wasn't supposed to give any band at that region... Now the chance of contamination as u suggested.. yep i thought of this too and just to rule it out i did a PCR using both GSP and anchor primer without using any template.. and as expected it gave no band.. so i ruled out the the idea of cross contamination of reagents or vials, etc.. i dont know how far i'm correct... may b ur suggestion again can shed some light on it... actually what i am trying to say is, its not like that the particular gene i'm trying to work on is not only present in the huge pool of cDNAs, many other cDNAs from different genes can be present in the pool.. but as per the assay result the possibility of cDNA from my gene of interest should be highest in the pool.. so along with the cDNA synthesis of my gene of interest there may be other genes too whose cDNA synthesis is taking place.. so is there's any chance that the GSP is finding some complementary region in some other cDNA where the same GSP is binding as the Forward and the Reverse Primer and giving away the spurious band at the desired position... if this is the chance can you please suggest me a better idea how to eradicate or reduce such type of spurious bands..??/... because if i use both the GSP and the anchor primer in combination with the cDNA template, the band which will be giving by them shall b highly ambiguous as the GSP itself only is giving away the same band at the same region.... Can u please suggest some ideas how to proceed with the GSPs without chance of getting those aforesaid spurious bands..??.. Is there any other mechanism by which i can check if there's any contamination playing the role..??... Thanxxxxxxxx.....
Reply With Quote
  #5  
Old 09-12-2009, 09:32 AM
ari ari is offline
Pipette Filler
Points: 75, Level: 1 Points: 75, Level: 1 Points: 75, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 6
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

Hiiiiiiiiiiii Jonoave.....

Thanxxx for replying....

...let me explain briefly how I am performing 3' RACE...
First of all i'm not using any RACE kit frrom Fermentas... i'm just using their cDNA synthesis kit comprising M-MuLV Reverse Transcriptase, RiboLockô RNase Inhibitor, 5X Reaction Buffer, dNTP Mix (10 mM each), Oligo(dT)18 Primer, DEPC treated water, etc. along with Control RNA and Control primers just to synthesize the cDNA from the total RNA extracted. Now, previously i used to synthesize cDNA using Oligo dT 27 Primer but last time it was synthesized using Oligo dT anchor primer. Using the resultant cDNA as the template i used to perform the 3' RACE using the Gene Specific Primer (GSP) as the forward primer and the anchor primer as the reverse primer. These GSPs have been designed from the conserved region of the same gene from the other organisms of same class maintaining stringent parameters. Now when I performed PCR using these GSPs and anchor primer, in three cases I got the band at the desired region .. But as I reported before, when i crosschecked using only the GSP with the template in one set and using only the anchor primer with template in another set it (GSP) gave band at the desired region which wasn't supposed to give any band at that region... Now the chance of contamination as u suggested.. yep I considered this too and just to rule it out i did a PCR using both GSP and anchor primer without using any template.. and as expected it gave no band.. so i ruled out the idea of cross contamination of reagents or vials, etc.. I donít know how far i'm correct... may b ur suggestion again can shed some light on it... actually what i am trying to say is, itís not like that the particular gene Iím trying to work on is not only present in the huge pool of cDNAs, many other cDNAs from different genes can be present in the pool.. but as per the assay result the possibility of my gene of interest should be highest in the pool.. so along with the cDNA synthesis of my gene of interest there may be other genes too whose cDNA synthesis is taking place.. so is there's any chance that the GSP is finding some complementary region in some other cDNA where the same GSP is binding as the Forward and the Reverse Primer and giving away the spurious band at the desired position... if this be the chance can you please suggest me a better how to eradicate or reduce such type of spurious bands..??/... because if I use both the GSP and the anchor primer in combination with the cDNA template, the band which will be giving by them shall b highly ambiguous as the GSP itself only is giving away the same band at the same region.... Can u please suggest some ideas how to proceed with the GSPs i have designed, bypassing all the confusions imposing by those spurious bands..??>Ö..
Reply With Quote
  #6  
Old 09-19-2009, 07:49 AM
ari ari is offline
Pipette Filler
Points: 75, Level: 1 Points: 75, Level: 1 Points: 75, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 6
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

?????????????????????????????????????????????????? ???????????..........
Reply With Quote
  #7  
Old 10-01-2009, 12:46 PM
Graduate Student
Points: 3,129, Level: 36 Points: 3,129, Level: 36 Points: 3,129, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2008
Posts: 108
Thanks: 4
Thanked 48 Times in 42 Posts
Default Re: Single primer giving spurious band at the desired position after 3' RACE ..

Sorry for the late reply.

As I mentioned before, I find it interesting that you are using a modified manual RACE protocol without a kit, just like another poster on this board. I'm not familiar with the method you're using, however after googling it I find that yours is a modified method from Roche's RACE kit technology.

I suggest you check out that manual, and try to compare the difference in the method between the kit and yours.
[Only registered users see links. ]
Reply With Quote
Reply

Tags
3' race , band , desired , giving , negative control , pcr troubleshooting , position , primer , race , single , spurious , spurious bands


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
Single primer giving spurious band at the desired position after 3' RACE .. ari PCR - Polymerase Chain Reaction Forum 0 09-08-2009 07:58 AM


All times are GMT. The time now is 08:07 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 0.16965 seconds with 16 queries