AlanFung:LabNotes/2015/2015-1-22: Difference between revisions
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*Place tray on an orbital shaker for ~5 min. | *Place tray on an orbital shaker for ~5 min. | ||
*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. | *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 (300-350 bp in this case), chop the band into small slices, | *Cut out the correct band (300-350 bp in this case), chop the band into small slices, pool two gel slices into one gel shearing assembly. | ||
*Change gloves and plastic wrap when size selecting a new gel. | *Change gloves and plastic wrap when size selecting a new gel. | ||
[[File:ZhangLab_2 2015-01-25 16hr 53min.jpg|400px]] | |||
[[File:ZhangLab_2 2015-01-25 16hr 51min.jpg|400px]] | |||
[[File:ZhangLab_2 2015-01-25 16hr 47min.jpg|400px]] | |||
[[File:ZhangLab_2 2015-01-25 18hr 28min.jpg|400px]] | |||
[[File:ZhangLab_2 2015-01-25 18hr 26min.jpg|400px]] | |||
[[File:ZhangLab_2 2015-01-25 18hr 24min.jpg|400px]] | |||
*Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. | *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. | *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. | ||
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*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. | *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. | *Let dry in hood ~ 10 min. | ||
*Add | *Add 25 uL of water per tube to resolve the DNA, leave at least 15 min. | ||
Store at 4C if needed, spin down before proceeding | Store at 4C if needed, spin down before proceeding | ||
=== Step 5. TBE Gel Size Verification === | === Step 5. TBE Gel Size Verification === | ||
===Step 6. Sequencing Prep=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| align="center" width="151" height="15" | | |||
| width="68" | nM | |||
| width="89" | To get 40nM | |||
| width="105" | Pooled Volume | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_6P Jan22 | |||
|style="background-color:#538DD5" align="center" | 34.87 | |||
|style="background-color:#538DD5" align="center" | 1.15 | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_7P Jan22 | |||
|style="background-color:#538DD5" align="center" | 19.85 | |||
|style="background-color:#538DD5" align="center" | 2.01 | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p1 Jan22 | |||
|style="background-color:#538DD5" align="center" | 26.08 | |||
|style="background-color:#538DD5" align="center" | 1.53 | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p2 Jan22 | |||
|style="background-color:#538DD5" align="center" | 12.23 | |||
|style="background-color:#538DD5" align="center" | 3.27 | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_NC.p3 Jan22 | |||
|style="background-color:#538DD5" align="center" | 34.25 | |||
|style="background-color:#538DD5" align="center" | 1.17 | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#538DD5" height="15" | HL-BSPP V5A_PCP Jan22 | |||
|style="background-color:#538DD5" align="center" | 25.59 | |||
|style="background-color:#538DD5" align="center" | 1.56 | |||
| align="center" | 10.70 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | HL-Seqcap pCancerSetA Jan5 | |||
| align="center" | 85.88 | |||
| n/a | |||
| n/a | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | HL-Seqcap pNormalSetA Jan5 | |||
| align="center" | 136.01 | |||
| n/a | |||
| n/a | |||
|} |
Latest revision as of 23:02, 6 February 2015
Size Selection of LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient[edit]
Background[edit]
- 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 300-350bp (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.
Part I: Gel Size Selection [edit]
Step 1. Pool libraries for size selection[edit]
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[edit]
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[edit]
- Run gel at 200V for 30mins
- Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
- Place tray on an orbital shaker for ~5 min.
- 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 (300-350 bp in this case), chop the band into small slices, pool two gel slices into one gel shearing assembly.
- Change gloves and plastic wrap when size selecting a new gel.
File:ZhangLab 2 2015-01-25 16hr 53min.jpg File:ZhangLab 2 2015-01-25 16hr 51min.jpg File:ZhangLab 2 2015-01-25 16hr 47min.jpg File:ZhangLab 2 2015-01-25 18hr 28min.jpg File:ZhangLab 2 2015-01-25 18hr 26min.jpg File:ZhangLab 2 2015-01-25 18hr 24min.jpg
- 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[edit]
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 25 uL of water per tube to resolve the DNA, leave at least 15 min.
Store at 4C if needed, spin down before proceeding
Step 5. TBE Gel Size Verification[edit]
Step 6. Sequencing Prep[edit]
nM | To get 40nM | Pooled Volume | |
HL-BSPP V5A_6P Jan22 | 34.87 | 1.15 | n/a |
HL-BSPP V5A_7P Jan22 | 19.85 | 2.01 | n/a |
HL-BSPP V5A_NC.p1 Jan22 | 26.08 | 1.53 | n/a |
HL-BSPP V5A_NC.p2 Jan22 | 12.23 | 3.27 | n/a |
HL-BSPP V5A_NC.p3 Jan22 | 34.25 | 1.17 | n/a |
HL-BSPP V5A_PCP Jan22 | 25.59 | 1.56 | 10.70 |
HL-Seqcap pCancerSetA Jan5 | 85.88 | n/a | n/a |
HL-Seqcap pNormalSetA Jan5 | 136.01 | n/a | n/a |