Thursday, February 5, 2015

2 5 2015 Bisulfite Conversion

Today I completed my second attempt at Bisulfite conversion of the previously sequenced samples. Last time the samples were sonicated which resulted in fragments too small for the conversion at least by the kits instructions. The samples themselves failed to take for library creation so I'm attempting again to produce libraries for sequencing. Here I used the newest kit in our collection to ensure the quality of the kit components.

Samples and Quantity of water used for BS Conversion to great 200 ng in 24 ul were the following:

Plate NumberD4B4G4H3E5D6G5G6H9D9A9G10
Volume (ul) for 200 ng2.
Water add (ul)21.421.321.521.320.319.921.619.720.420.120.420.2

The following was then performed based on the kits instructions:

  1. Added 1 ul of R1 to each sample
  2. Incubated sample for 10 minutes at 37C in moistened well heat block
  3. Made the R1/2/3 solution by adding 1.1 ml R3 to R2 powder and mixed till no solutes visible then added 40 ul of R1 solution. 
  4. Added 125 ul of fresh R1/2/3 solution to samples 1-10. Ran out but opted to use vial made up on 1/20/2015 by me instead of making more fresh solution since it has a short shelf life of around 2 weeks. The previously made vial is just past expiration but it was assumed it would still be viable. Added previously made solution to last two samples. 
  5. Vortexed samples vigorously and incubated at 65C for 90 minutes in moistened well heat block. 
  6. Added 300 ul R4 to sample, mixed through pipetting, and transferred to spin column in collection tube. 
  7. Centrifuged at 12000 rpm for 15 seconds (5 second spinup). Discarded flowthrough.
  8. Added 200 ul R5 solution (100% EtOH added previously to R5) to the column.
  9. Centrifuged 12,000 rpm 15 seconds (5 second spinup)
  10. Added 50 ul previously maded R1 solution (1.1 ml 90% EtOH with 10 ul R1) to 8 samples. Made fresh R1 solution and added it to the 4 remaining samples. 
  11. Incubated for 8 minutes at room temp.
  12. Centrifuged 12,000 rpm 15 seconds (5 second spinup) Discard Flowthrough
  13. Add 200 ul 90% EtOH to column
  14. Centrifuged 12,000 rpm 15 seconds (5 second spinup)
  15. Added 200 ul 90% EtOH to column
  16. Centrifuged 12,000 rpm for 35 seconds (5 second spinup) Discard flowthrough
  17. Placed columns in new 1.5 ml tubes.
  18. Added 18 ul of R6 to column filter.
  19. Centrifuged 12,000 rpm 20 seconds (5 second spinup)
  20. Stored samples at -20 C. 

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