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

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*Run gel at V for mins
*Run gel at V for mins
*Stain gel in new pipette lid with ul SYBR Gold  
*Stain gel in new pipette lid with ul SYBR Gold  
*Cut bands at  
*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. 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 05:45, 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. Gel Running & Size Selection Conditions

Step 5. TBE Gel Size Verification