Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > General Science Forums > Biochemistry Forum > DNA Forum
Register Search Today's Posts Mark Forums Read

DNA Forum Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


DNA extraction and PCR problems from bone

DNA extraction and PCR problems from bone - DNA Forum

DNA extraction and PCR problems from bone - Discuss DNA, the molecule of hereditary. Topics include DNA structure, DNA replication, DNA function.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 02-08-2009, 06:51 PM
Pipette Filler
Points: 61, Level: 1 Points: 61, Level: 1 Points: 61, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 3
Thanked 0 Times in 0 Posts
Unhappy DNA extraction and PCR problems from bone



Hello,
I was able to extract DNA from bone of a turtle species and see a week DNA band comparison to muscle tissue when I run the DNA products on the gel. I used Kalmar et al.,2000 protocol;
- Extraction buffer (0.1 M EDTA, 0.5% N-laurylsarcosinate, 100 mg/ml Proteinase K)
- 3.5 l (1 g/l) Dextran Blue
- 250 l 4 M NH4-acetate
- 500 l EtOH
- 25 l dH2O

I performed PCR of mtDNA gene(app. 850bp) to be optimized previously. But I didnt have amplification. I used in amplification;
- 0.6 l Primer F and R
- 2 l dNTP (2.5 mM)
- 1.5 l Bovin Serum Albumine (BSA) (4 g/ml)
- 0.4 l Taq poly.
- 6 l DNA (uncertain DNA quantitative)

I will be happy if you help me for both PCR and DNA extraction.
Thank you
Reply With Quote
  #2  
Old 02-09-2009, 11:07 AM
Pipette Filler
Points: 53, Level: 1 Points: 53, Level: 1 Points: 53, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 3
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: DNA extraction and PCR problems from bone

As per your protocol, may I ask you one question?
Didn't you use buffer and MgCl2 in your reaction mixture?
Reply With Quote
  #3  
Old 02-09-2009, 01:59 PM
Pipette Filler
Points: 61, Level: 1 Points: 61, Level: 1 Points: 61, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 3
Thanked 0 Times in 0 Posts
Default Re: DNA extraction and PCR problems from bone

I didn't write buffer and MgCl2 I used. I use 2.5 l/per 25 l reaction from 10X complete buffer.
Reply With Quote
  #4  
Old 02-10-2009, 02:03 PM
Pipette Filler
Points: 53, Level: 1 Points: 53, Level: 1 Points: 53, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 3
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: DNA extraction and PCR problems from bone

Hi,
You should try with different primers.
There could be possibility of presence of PCR inhibitors. Try to optimize DNA extraction protocol.
Reply With Quote
The Following User Says Thank You to Shraddha Bhullar For This Useful Post:
trionyx (03-09-2009)
  #5  
Old 02-11-2009, 06:54 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: DNA extraction and PCR problems from bone

pcr inhibitors can be check by including a know template and fully working primer pair in with the reaction you use. if that does not amplify then maybe it is due to inhibitors. since its mtDNA you cannot extract from other easy to obtain source? like fresh or blood?

u may need to quantify the amount and purity of ur extracted sample too. It may help to control the quality and concentration of DNA. Sometime too much DNA will inhibit PCR
Reply With Quote
The Following User Says Thank You to butters For This Useful Post:
trionyx (03-09-2009)
  #6  
Old 02-11-2009, 06:18 PM
Aga's Avatar
Aga Aga is offline
Post-Doc
Points: 2,239, Level: 30 Points: 2,239, Level: 30 Points: 2,239, Level: 30
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 270
Thanks: 25
Thanked 78 Times in 64 Posts
Default Re: DNA extraction and PCR problems from bone

As Butters said - you may have to check the quantity and purity of your DNA. If there's nothing - try to change something in your extraction protocol. I don't actually know how long do you incubate the samples during DNA extraction. You said there is a weak band on a gel so you have something but maybe too little.

If you have DNA after extraction the easiest way to get rid of high concentration of inhibitors is to dilute the sample. Have you tried your assay on other tissues? I worked with mtDNA and there is plenty of it in muscles - muscle cells need energy to work so there are many mitochondria.

Before doing PCR you need to have relatively pure DNA to optimize the reaction first. I don't suspect high amounts of mtDNA in bones. But if you extract some of DNA (most probably total DNA) from bones maybe the best solution would be to run nested PCR?
Reply With Quote
The Following User Says Thank You to Aga For This Useful Post:
trionyx (03-09-2009)
  #7  
Old 02-12-2009, 08:59 AM
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: DNA extraction and PCR problems from bone

can any body tell why dna has thymine in place of uracil ?
plz do tell me
Reply With Quote
  #8  
Old 02-12-2009, 08:59 AM
Pipette Filler
Points: 6, Level: 1 Points: 6, Level: 1 Points: 6, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: DNA extraction and PCR problems from bone

can any body tell why dna has thymine in place of uracil ?
plz do tell me
Reply With Quote
  #9  
Old 02-17-2009, 06:06 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: DNA extraction and PCR problems from bone

as far as i know uracil only available in RNA. correct me if i am wrong.
Reply With Quote
The Following User Says Thank You to butters For This Useful Post:
admin (02-17-2009)
  #10  
Old 07-23-2010, 08:37 PM
luisillo's Avatar
M.D/Ph.D
Points: 3,877, Level: 41 Points: 3,877, Level: 41 Points: 3,877, Level: 41
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2010
Location: Mexico
Posts: 352
Thanks: 19
Thanked 98 Times in 90 Posts
Thumbs up Re: DNA extraction and PCR problems from bone

Quote:
Originally Posted by vituljain View Post
can any body tell why dna has thymine in place of uracil ?
plz do tell me
It's basically a matter of stability. Thymine has its ring methylated. That methyl group confers more stability (less reactivity) to the molecule. This is pretty convinient since DNA acts as an "archive" of information and needs to be fidely conserved.

Another reason is the repair mechanisms of DNA replication: when a uracil is found in DNA it is converted to citosine by adding an amino group. This is why uracil normally never shows up in DNA, and cells most avoid this at all costs since it would mean a substitution of T for C.
Reply With Quote
Reply

Tags
bone , dna , dna extraction , extraction , pcr , problems


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


All times are GMT. The time now is 08:53 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 0.16898 seconds with 15 queries