Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Forum > Molecular Biology News and Announcements > Molecular Station Suggestion Forum
Register Search Today's Posts Mark Forums Read

Molecular Station Suggestion Forum Molecular Station Suggestion Forum. Post your suggestions, questions, and site comments here.


Help needed urgently

Help needed urgently - Molecular Station Suggestion Forum

Help needed urgently - Molecular Station Suggestion Forum. Post your suggestions, questions, and site comments here.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 08-31-2011, 04:15 PM
Pipette Filler
Points: 427, Level: 8 Points: 427, Level: 8 Points: 427, Level: 8
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 8
Thanks: 1
Thanked 3 Times in 3 Posts
Angry Help needed urgently



Hello,
I am currently working on amplifying a gene by nested PCR. The expected size in the first cycle is 1003bp and in the second cycle it is 483 bp. I was getting good results earlier but now am getting only smears with samples that were positive earlier. I have tried changing the reagents and also doing the pcr with fresh DNA, but no results.

I want to know if a gradient of annealing temperatures would help to optimise it again .I really do not have any more options . There was another doubt in my mind - Will amplification be seen after the first cycle in a nested PCR?

Desperately yours,
Nita
Reply With Quote
  #2  
Old 08-31-2011, 05:27 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: Help needed urgently

Quote:
Will amplification be seen after the first cycle in a nested PCR?
Can be either Yes or No--depends on the amount of target DNA in the sample, some don't become visible amounts until amplification by nested PCR.

I would repeat with the freshest DNA you have, make sure the thermocycle programs are all entered correctly, and primers are also fresh working solutions, might want to add a tiny extra amount of Taq as well.
Reply With Quote
The Following User Says Thank You to danfive For This Useful Post:
admin (09-06-2011)
  #3  
Old 08-31-2011, 05:31 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: Help needed urgently

Just noticed you mentioned smears--that can be RNA (smears are in the lane not necessarily the area of PCR product) rRNA is the primary culprit in smears, RNAse step or dilution helps) or DNA degradation in sample.
Reply With Quote
The Following User Says Thank You to danfive For This Useful Post:
admin (09-06-2011)
  #4  
Old 09-01-2011, 09:01 AM
Pipette Filler
Points: 427, Level: 8 Points: 427, Level: 8 Points: 427, Level: 8
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 8
Thanks: 1
Thanked 3 Times in 3 Posts
Default Re: Help needed urgently

Dear Dan,
Thanks for the suggestions. Will repeat with fresh samples.

Nita
Reply With Quote
The Following User Says Thank You to Nita Rajakumar For This Useful Post:
admin (09-06-2011)
  #5  
Old 09-01-2011, 03:39 PM
Pipette Filler
Points: 427, Level: 8 Points: 427, Level: 8 Points: 427, Level: 8
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 8
Thanks: 1
Thanked 3 Times in 3 Posts
Default Re: Help needed urgently

Thanks Danfive. I have checked for the quality of genomic DNA which was seen as a bright band of high molecular weight with no smears in the lane below.Is there a chance that RNA contamination can happen with the reagents tho I have tried changing all the reagents. I will try a dilutuon step too to reduce the smears.

Nita
Reply With Quote
The Following User Says Thank You to Nita Rajakumar For This Useful Post:
admin (09-06-2011)
Reply

Tags
needed , urgently


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
Research News - Pediatric-specific research needed to reduce health care-associated i moleculardude Science News and Views 0 04-21-2011 11:14 AM
How much plant biomass is needed per human? shoemakerted@yahoo.com Botany Forum 3 07-19-2007 03:17 PM
ELISA HELP NEEDED Cant Get anything to Work (Allison) Dan Guire Protein Forum 1 03-28-2007 08:27 PM
Percentage of sunlight needed by plants for photosynthesis? pchris@netcourrier.com Botany Forum 1 06-02-2006 09:11 PM
[Cell-biology] Looking for plasmid sequence - help needed! Jayakumar, R Cell Biology and Cell Culture 1 01-06-2006 07:15 PM


All times are GMT. The time now is 10:39 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 0.13896 seconds with 16 queries