Pac Bio Notes

PacBio library prep notes and protocols #

Malika lab notes #

PCB-04 folder #

  • Notes on footprinting “Set 2 PLB”.
  • Notes for prepping genomic DNA for footprinting.
  • PCR amplification of libraries using PhusionU pol with GC buffer for 30 cycles.
  • Includes PacBio 2kb Template Prep and Sequencing protocol but does not mention use of PacBio Template prep kit.
    • Assuming this kit is used though because no mention on making homemade reagents.
  • PCR follows bisulfite conversion reactions. After PCR preforms 1x Ampure bead wash.

In vitro footprinting folder #

This seems like what I am looking for. PacBio library prep notes related to pFC53 footprinting data.

  • Inculded PacBio 2kb template prep protocol with annotations

Genome center sample requirements #

Sample quantity #

Fragments will be around 3 kb so should include 5 ug of sample if delivered for library prep. For 33 plasmids (31 VR inserts, positive and negative controls) that is 0.161 ug or 161 ng of each plasmid. NEB recommends up to 1 ug DNA template per 50 ul T7 reaction.

So when completing actual protocol would use 1 ug total of all plasmids then split into 2 500 ng samples for bisulfite conversion as recommended DNA mass for the kit is 500 ng. After bisulfite conversion re-measure DNA concentration and PCR. Will need to determine the number of cycles in order to amplify to sufficient concentration. To me it would make sense to PCR a large amount of DNA in order to minimize duplicates.