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.


Taq Dilution

Taq Dilution - PCR - Polymerase Chain Reaction Forum

Taq Dilution - 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 09-02-2009, 05:21 PM
Pipette Filler
Points: 478, Level: 9 Points: 478, Level: 9 Points: 478, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 4
Thanks: 0
Thanked 1 Time in 1 Post
Default Taq Dilution



Hi,

I'd like to ask for instructions about Taq DNA polymerase dilution. To avoid pipetting errors I have to dilute Taq, which is supplied in a concentration of 5 units / microL, to a concentration of 0.5 units / microL . I just wonder if I can use PCR reaction buffer for the dilution process. I know Taq is supplied in a special buffer which contains Glycerol , surfactants , etc . and I would prefer to use a solution of similar composition for dilution but that needs to be purchased and there doesn't seem to be many suppliers for that anyway. So I wonder if I can make a 1:10 dilution using just the PCR buffer and store in aliquotes safely . I am highly concerned about storing Taq in just PCR buffer. I know I could just make a dilution, use the amount I need , then throw away the rest of the solution but I just can't afford that. I need to store the rest for future use.

Thank you in advance.
Reply With Quote
  #2  
Old 09-03-2009, 01:27 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: Taq Dilution

Hmm...I have yet to dilute my Taq DNA polymerase at all, but I don't see why you can't just dilute it in molecular grade water.

Let's see what the rest says.
Reply With Quote
  #3  
Old 09-03-2009, 04:39 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: Taq Dilution

I'm extremely curious to know what kind of PCR reaction you are doing that requires you to dilute your Taq. Taqs are generally supplied in 5U/ul, and you generally use less than 0.05 unit in a PCR reaction.

Eg: In a 50 ul reaction mix, add in 0.5 ul of Taq(5U/ul).
Whereby 5U x 0.5 ul / 50 ul = 0.05 units.

I usually use less than that, around 0.037 units with perfectly good results. However, if you do insist on diluting it, I would think it's best you ask the manufacturer/supplier if they have dilution buffers (I know one company which provides dilution buffers for their Pfu). If not, I think that it's better if you use the 10x buffer to dilute your Taq. Do it in small amounts in many aliquots to prevent using the same tube of diluted Taq repeatedly and undergoing thawing/freezing.
Reply With Quote
The Following User Says Thank You to jonoave For This Useful Post:
admin (09-04-2009)
  #4  
Old 09-03-2009, 09:04 PM
Pipette Filler
Points: 478, Level: 9 Points: 478, Level: 9 Points: 478, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 4
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Taq Dilution

Quote:
Originally Posted by jonoave View Post
I'm extremely curious to know what kind of PCR reaction you are doing that requires you to dilute your Taq. Taqs are generally supplied in 5U/ul, and you generally use less than 0.05 unit in a PCR reaction.

Eg: In a 50 ul reaction mix, add in 0.5 ul of Taq(5U/ul).
Whereby 5U x 0.5 ul / 50 ul = 0.05 units.

I usually use less than that, around 0.037 units with perfectly good results. However, if you do insist on diluting it, I would think it's best you ask the manufacturer/supplier if they have dilution buffers (I know one company which provides dilution buffers for their Pfu). If not, I think that it's better if you use the 10x buffer to dilute your Taq. Do it in small amounts in many aliquots to prevent using the same tube of diluted Taq repeatedly and undergoing thawing/freezing.
Nothing special at all, just a typical PCR.
If I add 0.5 ul to 50ul reaction volume then that means I am delivering 2.5U to the reaction volume . It's known that the optimal amount ( not concentration) ranges from 0.2 to 2 units . The number you calculated , which is 0.05 , is a concentration not an amount . And it's a rather high concentration for Taq . Too much Taq may result into smear in Gel electrophoresis due to amplification of non-specific products . 2.5 units / 50 ul may be acceptable , but , still , 0.5 ul is a small volume to deliver , especially that Taq is supplied in a solution containing 50% glycerol which means it has high viscosity which further complicates pipetting small volumes .
Reply With Quote
The Following User Says Thank You to Bassaml7 For This Useful Post:
jonoave (09-05-2009)
  #5  
Old 09-04-2009, 08:55 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: Taq Dilution

Hmm true, that is basically what I do too. I just add 0.5uL to get 0.05units if I need that amount. Have never done any dilutions for my Taq. You can take jonoave's suggestion of asking the supplier/manufacturer.
Reply With Quote
  #6  
Old 09-05-2009, 06:17 AM
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: Taq Dilution

Quote:
Originally Posted by Bassaml7 View Post
Nothing special at all, just a typical PCR.
If I add 0.5 ul to 50ul reaction volume then that means I am delivering 2.5U to the reaction volume . It's known that the optimal amount ( not concentration) ranges from 0.2 to 2 units . The number you calculated , which is 0.05 , is a concentration not an amount . And it's a rather high concentration for Taq . Too much Taq may result into smear in Gel electrophoresis due to amplification of non-specific products . 2.5 units / 50 ul may be acceptable , but , still , 0.5 ul is a small volume to deliver , especially that Taq is supplied in a solution containing 50% glycerol which means it has high viscosity which further complicates pipetting small volumes .
Thanks for this! I knew there was something I was missing when typing my response, but I couldn't remember what it was. Yeah, Taq is usually quoted in units and not concentration. My bad...

Taq is highly sensitive; the one I'm using was prescribed as 1-2.5 units. I tend to use less the lower range, which is around 1.5 units. For visualisation, I use 0.1 ul of Taq for a 15 ul reaction and have consistent results.

As for pipetting Taq, I generally put it in the Mastermix, whereby I'll pippette a larger volume. I usually leave out the dNTPs or template out of the Mastermix, which is generally in a higher amount than Taq and is easier to pipette individually. I've never had any problems with this, and I find that Taq is hardly a factor in any failed PCRs.

Erm, so basically I just think that diluting Taq is not a good idea... :P
Reply With Quote
  #7  
Old 09-06-2009, 09:43 PM
Pipette Filler
Points: 478, Level: 9 Points: 478, Level: 9 Points: 478, Level: 9
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Posts: 4
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Taq Dilution

Quote:
Originally Posted by jonoave View Post
Thanks for this! I knew there was something I was missing when typing my response, but I couldn't remember what it was. Yeah, Taq is usually quoted in units and not concentration. My bad...

Taq is highly sensitive; the one I'm using was prescribed as 1-2.5 units. I tend to use less the lower range, which is around 1.5 units. For visualisation, I use 0.1 ul of Taq for a 15 ul reaction and have consistent results.

As for pipetting Taq, I generally put it in the Mastermix, whereby I'll pippette a larger volume. I usually leave out the dNTPs or template out of the Mastermix, which is generally in a higher amount than Taq and is easier to pipette individually. I've never had any problems with this, and I find that Taq is hardly a factor in any failed PCRs.

Erm, so basically I just think that diluting Taq is not a good idea... :P
I see, so I think preparing Mastermixes is the best practise . Thank you for your response .
Reply With Quote
  #8  
Old 09-06-2009, 10:06 PM
jyaron's Avatar
Pipette Filler
Points: 66, Level: 1 Points: 66, Level: 1 Points: 66, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2009
Location: Tempe, Arizona
Posts: 9
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Taq Dilution

Mastermixes is generally what my lab does.
Reply With Quote
Reply

Tags
dilution , taq


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
laemmli buffer dilution raya Western Blot Forum 3 09-23-2009 04:39 PM
Homéopathie : Dilution korsakovienne contre dilution Hahnemannienne Julien Arlandis Forum De Chimie 161 06-14-2008 11:36 AM
Dilution factor!!! euge Protocols and Methods Forum 2 10-15-2007 12:06 AM
Dilution cloning of HEK293 Neil Fraser Protocols and Methods Forum 1 04-27-2004 10:27 PM
Effect of Phosphate Dilution on pH N. Ron Chemistry Forum 5 11-20-2003 05:40 PM


All times are GMT. The time now is 07:01 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16645 seconds with 16 queries