Guidelines - DNA input

[lilletine]

Hello - I am very new to this, our lab has just set up til 5500xl and we are about to start the first run. I am looking all over for general guidelines regarding library DNA input for the ePCR but i am unable to find documentation for this other than the 'ePCR calculator' which suggests 500 pM input DNA. We learn that it is really important to get the correct ratio of DNA and beads so i would like to see recommendations in writing. I am very grateful for any help regarding this!

[JPC]

Hi, the ePCR calculator is the only way to calculate your input DNA. For an E120 reaction we use a Titration point of 0.616 picomoles (which you get my adjusting the "Desired P2% post enrich" to 18) the enter your library size and concentration (ideally around 70-80 pg/ul). Ultimately for an E120 we end up loading around 5,000 - 6,000 pg of DNA depending on your product size.

In terms of general advice, we normally use with 2ug of DNA to make a library, we found using the max (5ug) often caused problems, as did other users we know. Also after you have ligated your adaptors you come to an 'optional' amplification step.... we have found that this is NOT optional. No matter how much library you have at this stage I recommend doing the number of cycles listed in the table (page 26 of the Frag Lib prep User Guide).

Good luck with your run

JC

[JPC]

also, I'm not sure if it says this in the manuals but we tend to try and load 400M beads per lane

JC

[lilletine]

Thank you very much for your help!

[yhey]

James, I am interested to know why you think the optional amplification is not optional. What problems have you encountered by not amplifying?

Thanks
Yvonne

[JPC]

Hi Yvonne, skipping this step was the cause of the problems we experienced at the start (with the Enricher not returning the beads we were expecting). AB suggested that at the end of library prep there is still a significant proportion of fragments with no adaptor or just 1 adaptor so the 'optional amp' enriches for the fragments with 2 adaptors (P1-P2, P1-P1 and P2-P2).

[yhey]

Hi Yvonne, skipping this step was the cause of the problems we experienced at the start (with the Enricher not returning the beads we were expecting). AB suggested that at the end of library prep there is still a significant proportion of fragments with no adaptor or just 1 adaptor so the 'optional amp' enriches for the fragments with 2 adaptors (P1-P2, P1-P1 and P2-P2).

OK I thought that might be your reply. I have just had a similar experience with my first unamplified fragment libraries in that an E80 enrichment gave a bead yield less than expected from E20 but the sequence quality looks fine. I'll definitely amplify next time though!

Thanks for the post James.