Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

PCR triplicates versus one reaction


  • Please log in to reply
17 replies to this topic

#16 molbio1234

molbio1234

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
2
Neutral

Posted 27 March 2011 - 05:08 PM

I am getting the feeling my question in the above post is more of a trial and error, which is a pain :blink:. Does anyone have any guidance or suggestions ?

#17 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 28 March 2011 - 09:33 PM

No reference this time.

Or if your research was really tricky and you had to put in x number of copies of your DNA, then how to go about figuring out what x is to amplify your target correctly and avoid PCR bias.

If you know your gene sequence, design a primer. If you can extract and purified your DNA, make it in 10pg concentration. Do a 10x dilutions till 1fg level.
Run your PCR in gradient.

You have to do all the above (unless I missed out something...) in order to answer your question. You will know the minimum template DNA required for that particular PCR to work. No general or common or fixed number of amount.

I not sure what you mean by PCR bias. Bad primer design: unspecific amplification.


the effect of a low number of copies of DNA to put into your PCR.

too low maybe no amplification
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#18 Maddie

Maddie

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 289 posts
3
Neutral

Posted 31 March 2011 - 07:36 AM

you could also do one amp in 100ul (basically, you dilute your DNA without decreasing the amount of starting template).


i don't understand how you can dilute the dna without decreasing the starting template amount. can you give some examples.



If you put 50ng in 50ul, you have a concentration of 1ng/ul. If you put 50ng in 100ul, you have a concentration of 0.5ng/ul but you still have 50ng in your tube.

As for the low copy number PCR, we usually increase the number of cycles in order to detect PCR product on a gel.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.