Matt:LabNotes/2013-6-26

From ZhangLabWiki
Jump to navigation Jump to search

Page Size Selection of 0 gap probes[edit]

USER/DpnII digested 0 gap probes:

 Noi made 11.36ug/~400ul 2013-5-29 
 I made 10.58ug/180ul 2013-5-16 

To run ~3ug/gel, I use ~100ul of probes that Noi made for each of the 4 gels

  • Ran TBU gel at 220V for 30 min with samples and ladder prepared below
    • Heated TBE buffer in microwave
    • Denatured samples/ladder for 10 min at 75C before putting on cold rack for 1 min
    • Size selected for brightest band below 200bp
Components 1X Volume 4X Volume
Enzyme digested probes (~11.36/400ul) 106 424
TBE-Urea Buffer 2X 106 424
H2O 0 0
Total 212 848
Components 1X Volume 4X Volume
Low MW DNA ladder (1 ug/ul) 1 4
TBE-Urea Buffer 2X 5 20
H2O 4 16
total 10 40

File:2013-06-26 Agi26k 0gap SizeSelect1.jpg File:2013-06-26 Agi26k 0gap SizeSelect2.jpg File:2013-06-26 Agi26k 0gap SizeSelect3.jpg File:2013-06-26 Agi26k 0gap SizeSelect4.jpg

  • Put cut out gels in 0.5mL tube with holes at bottom in 2 mL tubes (6 tubes for 4 gels)
  • Centrifuged the tubes at 15,000 rpm, for 3 min at RT.
  • Transfered the gel remaining in 0.5 mL tube to 2 mL tube below with pipette tip.
  • Added 675 ul of 1X TE buffer (because 1.5x more gel compared to normal protocol using 1.5mL tube)
  • Vortexed for 45-60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to fresh 1.5 mL tube (volume ~ 500ul/ tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C overnight (had to use 8 because volume was too much for 6)

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-27

First Strand cDNA synthesis[edit]

  • Followed NEB E6300S protocol: [1]

Got 2ul aliquot of Human Brain Reference RNA from Blue at concentration 1ug/ul

  • Mixed components in two 0.2 ml microfuge tubes labelled HBBR 1ug cDNA
Components Volume 2X Volume
RNA 1 ul (1 ug) 2 ul (2 ug)
d(T)23VN (50uM) 2 ul 4 ul
H2O 5 ul 10 ul
Total 8 ul 16 ul
  • Denatured RNA for 5 min at 70C and then put on ice
    • Tightened the thermocycler too much and bent the tube slightly out of shape
  • Added the following to each tube
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Kept on ice

Second Strand cDNA synthesis[edit]

  • Follow NEB E6111S protocol: [2]
  • Added 48 ul H20 to each tube
  • Added 8ul 10x Second strand synthesis reaction buffer
  • Added 4ul Second strand synthesis enzyme mix
  • Mixed by pipetting
  • Incubated for 2.5 hours at 16C
  • Purified using Qiagen minelute column (eluted 23ul into 1.5ml tube labelled HBRR cDNA)
  • Measured DNA conc with Nandrop:
    • 43ul at 63.7ng/ul (2.74 ug) sample left in each tube
    • "High yield" because RNA was not digested and went from single stranded to double stranded; slightly higher but consistent with last time 2013-3-9