AlanFung:LabNotes/2015/2015-1-22: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 462: Line 462:
*Spin down all tubes at 13,000rpm for 2min  
*Spin down all tubes at 13,000rpm for 2min  
*In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
*In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
===Step 4. Precipitation===
'''Turn on centrifuge let it cool down to 4C'''
*Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
*Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
*Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
*Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
*Let dry in hood ~ 10 min.
*Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding


=== Step 4. Gel Running & Size Selection Conditions ===
=== Step 4. Gel Running & Size Selection Conditions ===
=== Step 5. TBE Gel Size Verification ===
=== Step 5. TBE Gel Size Verification ===

Revision as of 05:47, 23 January 2015

Size Selection of LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient

Background

  • Kun thinks the results from the experiment Noi did on Illumina’s plasma samples looks promising.
  • He thinks we should repeat the same experiments on all our cancer and plasma samples.
  • We should have all those WGBS libraries ready, so it would be just a BSPP capturing experiment (we might need to make more probes though).
  • Kun asked Noi to help me identify the reagents and design a capturing experiment on all WGBS libraries
  • Noi said she had already finished the BSPP capture on WGBS libraries and she will need my help to pool the libraries and do size selection
  • Noi will update her wiki so I can follow her work
  • Noi used 600ng of WGBS libraries for capture instead of the 400ng for illumina.
  • She reduced the probe to target ratio from 200:1 to 125:1due to limited amount of probes.
  • She also used MONOD V5A probe set (LMS cluster) and the rest of the protocol are pretty much the same.
  • She quantified the library concentration by PAGE quantification, there seems to be a little high variation between different gels
  • She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
  • I will have to cut the get in the range between 300-350np (Do not of over 400bp) since she cropped the images to show me.
  • There will probably be high background over 400bp, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
  • Use one 5-well TBE gel per one pool of libraries
  • The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
  • Noi already filled in the sequencing spreadsheet
  • For Illumina experiment, Noi suggested Li to only pool sequencing libraries and re-purified with 0.7% AMPure beads to concentrate the libraries and remove the remaining primer dimers.
  • However, for Li libraries, I got low background of a smear over 400bp.

http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-12-16#PAGE_size_selection_.26_Sequencing_library_information

Part I: Gel Size Selection

Step 1. Pool libraries for size selection

Tube ID: NP-BSPP_V5A_NC.p1-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-NC-1_41 NC-1 5.23 13.39
NP-BSPP-V5A-Dec16-NC-2_42 NC-2 5.42 12.92
NP-BSPP-V5A-Dec16-NC-3_43 NC-3 9.44 7.42
NP-BSPP-V5A-Dec16-NC-5_44 NC-5 4.74 14.77
NP-BSPP-V5A-Dec16-NC-6_45 NC-6 5.54 12.64
NP-BSPP-V5A-Dec16-NC-7_46 NC-7 4.44 15.76
NP-BSPP-V5A-Dec16-NC-8_47 NC-8 4.01 17.46
NP-BSPP-V5A-Dec16-NC-9_48 NC-9 5.49 12.76
       
Tube ID: NP-BSPP_V5A_NC.p2-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-NC-12_49 NC-12 2.41 29.09
NP-BSPP-V5A-Dec16-NC-13_50 NC-13 2.14 32.66
NP-BSPP-V5A-Dec16-NC-14_51 NC-14 2.7 25.91
NP-BSPP-V5A-Dec16-NC-15_52 NC-15 2.65 26.45
NP-BSPP-V5A-Dec16-NC-16_53 NC-16 2.6 26.95
NP-BSPP-V5A-Dec16-NC-17_54 NC-17 3.37 20.8
NP-BSPP-V5A-Dec16-NC-18_55 NC-18 2.26 31.01
NP-BSPP-V5A-Dec16-NC-19_56 NC-19 3.55 19.72
       
Tube ID: NP-BSPP_V5A_NC.p3-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-NC-20_57 NC-20 5.5 18.17
NP-BSPP-V5A-Dec16-NC-21_58 NC-21 6.45 15.52
NP-BSPP-V5A-Dec16-NC-22_59 NC-22 6.02 16.61
NP-BSPP-V5A-Dec16-NC-23_60 NC-23 6.1 16.38
NP-BSPP-V5A-Dec16-NC-25_61 NC-25 5.68 17.61
NP-BSPP-V5A-Dec16-NC-27_62 NC-27 5.85 17.08
NP-BSPP-V5A-Dec16-NC-29_63 NC-29 5.82 17.18
NP-BSPP-V5A-Dec16-NC-30_64 NC-30 7.19 13.9
       
Tube ID: NP-BSPP_V5A_PCP-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-PCP-2_65 PCP-2 3.06 22.9
NP-BSPP-V5A-Dec16-PCP-3_66 PCP-3 2.32 30.13
NP-BSPP-V5A-Dec16-PCP-4_67 PCP-4 2.55 27.46
NP-BSPP-V5A-Dec16-PCP-5_68 PCP-5 3.56 19.65
NP-BSPP-V5A-Dec16-PCP-6_69 PCP-6 1.99 35.16
NP-BSPP-V5A-Dec16-PCP-7_70 PCP-7 2.25 31.16
NP-BSPP-V5A-Dec16-PCP-8_71 PCP-8 2.07 33.81
NP-BSPP-V5A-Dec16-PCP-9_72 PCP-9 4.03 17.36
       
Tube ID: NP-BSPP_V5A_6P-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-6P-1_73 6P-1 4.68 17.08
NP-BSPP-V5A-Dec16-6P-2_74 6P-2 3.99 20.05
NP-BSPP-V5A-Dec16-6P-3_75 6P-3 6.79 11.77
NP-BSPP-V5A-Dec16-6P-4_76 6P-4 4.62 17.31
NP-BSPP-V5A-Dec16-6P-5_77 6P-5 4.63 17.26
NP-BSPP-V5A-Dec16-6P-8_78 6P-8 5.58 14.35
NP-BSPP-V5A-Dec16-6P-9_79 6P-9 3.99 20.05
NP-BSPP-V5A-Dec16-6P-10_80 6P-10 4.87 16.42
       
Tube ID: NP-BSPP_V5A_7P-Dec16
Samples Name Sample ID Concentration (ng/ul) Volume for 70ng (ul)
NP-BSPP-V5A-Dec16-7P-1_81 7P-1 3.42 20.48
NP-BSPP-V5A-Dec16-7P-2_82 7P-2 1.6 43.62
NP-BSPP-V5A-Dec16-7P-3_83 7P-3 3.86 18.13
NP-BSPP-V5A-Dec16-7P-5_84 7P-5 3.22 21.74
NP-BSPP-V5A-Dec16-7P-6_85 7P-6 3.25 21.54
NP-BSPP-V5A-Dec16-7P-7_86 7P-7 4.18 16.75
NP-BSPP-V5A-Dec16-7P-8_87 7P-8 3.48 20.13
NP-BSPP-V5A-Dec16-7P-10_88 7P-10 3.69 18.98

Step 2. Add Gel Loading Dye and Load Gel

Sample ID Total 6X Dye Sum Per Well (4x)
Tube ID: NP-BSPP_V5A_NC.p1-Dec16 107.12 22 129.12 32.28
Tube ID: NP-BSPP_V5A_NC.p2-Dec16 212.59 45 257.59 64.4
Tube ID: NP-BSPP_V5A_NC.p3-Dec16 132.44 30 162.44 27.07
Tube ID: NP-BSPP_V5A_PCP-Dec16 217.63 45 262.63 65.66
Tube ID: NP-BSPP_V5A_6P-Dec16 134.3 30 164.3 41.07
Tube ID: NP-BSPP_V5A_7P-Dec16 181.37 40 221.37 55.34

Step 3. Gel Running & Size Selection Conditions

  • Run gel at V for mins
  • Stain gel in new pipette lid with ul SYBR Gold
  • Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
  • Cut out the correct band ( bp in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
  • Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
  • Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
  • Spin down all tubes at 13,000rpm for 2min
  • In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.

Step 4. Precipitation

Turn on centrifuge let it cool down to 4C

  • Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
  • Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
  • Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
  • Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
  • Let dry in hood ~ 10 min.
  • Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.

Store at 4C if needed, spin down before proceeding

Step 4. Gel Running & Size Selection Conditions

Step 5. TBE Gel Size Verification