AlanFung:LabNotes/CTCF/2011-8-26: Difference between revisions

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*the ones with more streaking failed
*the ones with more streaking failed
==Continue on 9/4==
==Continue on 9/4==
HF02
{| {{table}}
AGATGTGTATAAGAGACAGAAAGACCACTAAAGATAGTGTAACAAATGTCAAAGATGTAGTTGCCACTGGCCATTTCAAATCATGATGATTATCATCAGAGGCCAAAATGTAATGATGCCCTATAAGAGGATGTAGGGTTTATGTAATCTTGTGTGGACAATTTTCAGAGTTAATATCCTGTCTCTTATACGCATCT
| align="center" style="background:#f0f0f0;"|'''Expected Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Sequence'''
|-
| M13R||Orange Primer||Sample Sequence||Blue Primer||M13F
|-
| CAGGAAACAGCTATGAC||CCTTGCCAGCCCGCTCAG||N/A||CCTCCCTCGCGCCATCAG||GTAAAACGACGGCCAG
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Sequence'''
|-
| M13R||Orange Primer||Sample Sequence||Blue Primer||M13F
|-
| ||GACTCGCCCGACCGTTCC||N/A||GACTACCGCGCTCCCTCC||GACCGGCAGCAAAATG
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Complement Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Complement Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Complement Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Complement Sequence'''
| align="center" style="background:#f0f0f0;"|'''Expected Reverse Complement Sequence'''
|-
| M13R||Orange Primer||Sample Sequence||Blue Primer||M13F
|-
| ||CTGAGCGGGCTGGCAAGG||N/A||CTGATGGCGCGAGGGAGG||CTGGCCGTCGTTTTAC
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sequence 1'''
| align="center" style="background:#f0f0f0;"|'''Sequence 1'''
| align="center" style="background:#f0f0f0;"|'''Sequence 1'''
| align="center" style="background:#f0f0f0;"|'''Sequence 1'''
| align="center" style="background:#f0f0f0;"|'''Sequence 1'''
|-
| M13R||Orange Primer||Sample Sequence||Blue Primer||M13F
|-
| Sequence||CTGAGCGGGC||363bp||CCTCCCTCGCGCCATCAG||N/A
|-
| Location||364-373||124-363||106-123||N/A
|-
| Number of||A||T||C||G
|-
| #||131||78||82||64
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sequence 2'''
| align="center" style="background:#f0f0f0;"|'''Sequence 2'''
| align="center" style="background:#f0f0f0;"|'''Sequence 2'''
| align="center" style="background:#f0f0f0;"|'''Sequence 2'''
| align="center" style="background:#f0f0f0;"|'''Sequence 2'''
|-
| M13R||Orange Primer||Sample Sequence||Blue Primer||M13F
|-
| Sequence||CTGAGCGGGCTGGCAAGG||196bp||CCTCCCTCGCGCCATCAG||CTGGCCGTC
|-
| Location||304-321||107-303||89-106||633-641
|-
| Number of||A||T||C||G
|-
| #||67||59||30||41
|}
Sequence 3 Sequence 3 Sequence 3 Sequence 3 Sequence 3
M13R Orange Primer Sample Sequence Blue Primer M13F
Sequence CTGAGCGGGCTGGCAAGG 293bp CCTCCCTCGCGCCATCAG CTGGCCGTCGTTTTAC
Location 294-311 124-293 106-123 405-420
Number of A T C G
# 102 68 80 41
==Results & Improvements==
*Athurva and I both failed to generate bisulfite converted libraries. Could be due to human error while preparing the bisulfite conversion reagent (it was a new tube prepared by me), the protocol was simple and I am sure I didn't make mistake at that time, but I guess I will pay more attention when repeating the protocol.
*Ordered new Imprint kit and we will test it out
*Lyse cells with Naoh instead of NP40
*Perform Etoh precipitation instead of column after MDA

Latest revision as of 00:28, 20 September 2011

Low-input Bisulfite Conversion and MDA Nextera library[edit]

Protocols Today[edit]

  • Start with four tubes: 314 cells, 157 cells x 2, NTC all go through bisulfite conversion
  • Make 1571 cell/ul (original tube)
  • Make 314 cells/ul (dilute 10ul cells in 40 ul ddh2o)
  • Make 157cells/ul (dilute 10ul cells in 90 ul ddh2o)

Cell Lysis[edit]

  • First, make protease Dilution
    • Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
  • Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Cells are now lysed

Make New BSA-Water solution[edit]

  • Stock solution is at 10mg/ml, need to dilute to 0.5mg/ml
  • Take 10ul stock BSA dilute with 190ul nuclease free water

Bisulfite Conversion - Imprint Kit[edit]

  • Prepare DNA Modification Solution (CT Conversion Reagent):
  • Add 21 uL 0.5 mg/mL BSA and
  • Add 1 uL Imprint Balance solution to each sample;
  • incubate at 37 C for 10 minutes
  • Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
  • Perform Column Purification of each sample:
  • Elute into 11uL Elution Buffer

Continue on 8/28[edit]

  • Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content Volume MM
10X Repli Phi Buffer 2 8.8
1mM N9 Primer 1 4.4
25mM dNTP 0.8 3.52
Repliphi Phi 29 (100U/ul) 1 4.4
Klenow Exo Minus (10U/ul) 0.5 2.2
Nuclease free H20 4.7 20.68
Modified DNA Sample 10 Do not add to MM
Total 20 44
  • Add 10ul of MDA mastermix to each sample
  • 30C 60 min, 65C 10 min, 4C hold

MinElute[edit]

  • Elute with 10ul nuclease free water

Speedvac[edit]

  • To get 3ul for shendure's method of nextera library tagmentation, I will transfer the 10ul samples to qpcr tube, put the cap on and puncture a hole at the cap and speedvac till the sample is dry
  • took about 28min to dry samples
  • Re-dissolve the samples with 3ul of nuclease free water, sit at RT for 5 min

Tagmentation[edit]

  • To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes
  • To make 1:100 Protease, mix 1ul of protease with 99ul of nuclease free water
  • Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
  • Prepare mastermix for library amplification
Content Volume MM
Nuclease Free H2O 16 70.4
10uM Orange Primer 1 4.4
10uM Blue Primer 1 4.4
Tagmented DNA 6 Do not add to MM
2X Kapa SYBR Supermix 25 110
BST 1 4.4
Total 50 193.6
  • Add 44ul MM to each reaction
  • Perform qPCR using the following reaction conditions:
    • 65 C, 3 min incubation (BST gap filling)
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extension)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C
  • 300 cells was taken out after 7 cycles
  • 150 cells & NTC was taken out after 12 cycles

File:ZhangLab 2 2011-08-28.jpg

Purification[edit]

  • Minelute column, elute with 15ul water
  • Nanodrop gave me weird results, I was having problem when measuring the blank sterile water. Will avoid using the water provided by the Repliphi phi 29 kit for elution next time
    • 150:6.6ng/ul
    • 300:-0.2ng/ul
  • Use Qubit to measure the concentration instead
    • 150:4.16ng/ul
    • 300:1.46ng/ul
  • Looks like I do not have enough samples for end repair, may be I should run a few more cycles of PCR?
  • I would like to run a gel to confirm the size of the amplicons

Continue on 8/29[edit]

  • Talk to Dr. Zhang and he suggests that I can take 0.5ul of amplified samples and repeat the PCR, doing that will avoid over amplify all the samples.
  • Also me and Athurva need to come up with a plan on experiments to obtain a few aspects that we need to validate
    • whether bisulfite conversion is complete
    • whether the methylation level is accurately capture in the libraries
    • whether the resulting amplicons contain the right adaptors such that they can be sequenced on the illumina platform
    • whether all genomic regions are present at roughly equal abundance in the libraries
    • What can you get from the Sanger sequencing experiment, and what other experiments you need to do

Library Amplification[edit]

Content Volume MM
Nuclease Free H2O 22.5 74.25
10uM Orange Primer 1 3.3
10uM Blue Primer 1 3.3
Purified Amplicons 0.5 Do not add to MM
2X Kapa SYBR Supermix 25 82.5
Total 50 165
  • Add 49.5ul MM to each reaction
  • Perform qPCR using the following reaction conditions:
    • 95 C, 30 seconds (Denature)
    • 35 cycles of:
      • 95 C, 10 seconds (Denature)
      • 58 C, 30 seconds (Anneal)
      • 72 C, 3 minutes (Extension)
    • 72 C, 5 minutes (final extension)
    • Hold at 4 C

Minelute[edit]

  • Minelute with 15ul EB buffer
  • Nanodrop Result
    • NTC:25.9ng/ul
    • 150:94.1ng/ul
    • 300:85.5ng/ul

PAGE Gel Analysis[edit]

File:ZhangLab 2 2011-08-29 18hr 51min.jpg

Continue on 8/30[edit]

PAGE Size Selection[edit]

  • Talked to Noi, she suggests that I can load ~500ng/well for my sample size is a a big range, I will load 2 wells per sample
  • Load a 5well TBE gel into the gel running module
  • Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
Mix 1ul low mass ladder with 10ul dH2O, 9ul 6X loading dye
Mix 6.5ul samples with 4.5ul dH2O, 9 ul 6X loading dye 
  • Load the samples into the wells, run at 250V for 25 min.
  • Stain gel with 2.5uL SYBR Gold in ~100 mL of TBE in clean tray.
  • Place tray on an orbital shaker for ~5 min.

Purify the product from the gel[edit]

  • 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 (175-800 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.

File:ZhangLab 2 2011-08-30 15hr 04min.jpg

  • Centrifuge at top speed > 13000 rcf 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
  • Spin down all tubes at 13,000rcf 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.

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. (35ul 3M NAoAC, 875ul 100% ETOH)
  • 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 5 uL of water to each tube to resolve the DNA , leave at least 15 min.
  • Combine all 4 tubes into one

Qubit Measurement[edit]

    • 150cells:6ng/ul
    • 300cells:10.7ng/ul

Cloning for Sequencing[edit]

Blunting with NEB end repair kit[edit]

  • Take 8.5ul of PCR product
Content 150 300
Fragmented DNA 8.5 8.5
10X End repair reaction buffer 1 1
End reapir enzyme 0.5 0.5
H2O 0 0
Total Volume 10 10
  • Incubate at 20C for 30min
  • Minelute with 10ul EB

Qubit[edit]

    • 150cells:2.84ng/ul
    • 300cells:5.12ng/ul

Ligating into pCR blunt vector[edit]

    • Calculate the amount of PCR product needed for ligation
  • Talked to Rui and she used 5ng vector, I will try the same protocol as I got too many colonies from the previous cloning, however I will maintain insert to vector ratio at 10:1
    • x ng insert = (10*500*5/3500)
    • X=7.14ng needed
  • Make 5ng/ul vector (Mix 2ul vector with 8ul water)
Content 150 cells 300 cells
pCR-Blunt (5ng/ul) 1 1
Blunt PCR Product 2.51 1.39
10X ligation buffer (with ATP) 1 1
Sterile water 4.49 5.61
T4 DNA Ligase (4U/ul) 1 1
Total Volume 10.00 10.00
  • Incubate at 16C for 1 hr

Heat-shock transfection[edit]

  • Equilibrate water bath to 42C.
  • Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
  • Thaw the TOP10 E.coli competent cell vial on ice and split 25ul to a new 1.5ml tube
  • Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocl).
  • Set up conpetent cell vials in the foam-board holder(for water bath). Incubate the vials on ice for 30 mins.
  • Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
  • Add 125ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall).
  • Shake the vials horizontally at 37C for 1 hour using vortex in the incubator at the lowest speed.
  • During the 1hr incubation.
  • After 1hr shacking mixing, plate 50uL from each transformation vial on agar plates (w/ Kanamycine and xgal)
  • Incubate plates at 37C for about 20hr.

Continue on 8/31[edit]

  • After 15 hours incubation, there are no observable blue colored clones on both plates (this is unusually, we usually get some blue colonies).
  • Colonies are well spread out also, it was much better than last cloning, there are some spots that are over crowded, mostly due to uneven spreading
  • Colonies are a bit small will let them grow for a couple more hours

Continue on 9/1[edit]

  • Randomly pick colonies and inoculate into 10ul of dH2O
  • Heat denature the colony suspension at 94C for 10min, quickly transfer to cold block
Content Volume per Reaction MM
Template 2 Do not add to MM
Nuclease Free Water 8.5 69.7
M13 F (200ng) 1 8.2
M13 R (200ng) 1 8.2
Taq2X 12.5 102.5
Total 25 188.6
  • Aliquot 2ul of samples to a new 0.2ml tube
  • Add 23 ul MM to each sample
  • Vortex and spin down
  • Perform the following reaction in a thermo cycler
    • 95C 2min
    • 95C 30sec
    • 60C 30sec
    • 72C 1min
    • Go to step 2 for 29 cycles
    • 72C 5min
    • Hold at 4C

Page Gel Analysis[edit]

File:ZhangLab 2 2011-09-01 14hr 58min.jpg

  • It is abnormal to have streaking, it could be be to contamination from other colones, or just artifacts, but the sample amplicons (~600-800bp) are the dominant. Move on with purification and send out for sanger sequencing
  • Size of pCR Blunt vector is 270bp size of libraries are (175-800bp), so expected amplicons size are 445-1070bp, we are seeing amplicons between 600-800bp, it looks like we have the right size of amplicons
Label Content ng/ul 20ng dh2o 5uM M13R
HL01 150-1 7.2 2.78 7.22 5
HL02 150-2 7 2.86 7.14 5
HL03 150-3 8.5 2.35 7.65 5
HL04 150-4 9.8 2.04 7.96 5
HL05 300-1 9.5 2.11 7.89 5
HL06 300-2 5.9 3.39 6.61 5
HL07 300-3 9.3 2.15 7.85 5
HL08 300-4 6.7 2.99 7.01 5

Continue on 9/2[edit]

Sanger Sequencing Results[edit]

Label Content QS CRL Failure Cause
HF01 150-1 31 361 N/A
HF02 150-2 43 632 N/A
HF03 150-3 35 445 N/A
HF04 150-4 17 77 Early Termination
HF05 300-1 23 139 Early Termination
HF06 300-2 15 66 Poor Quality
HF07 300-3 32 261 Early Termination
HF08 300-4 43 429 N/A
  • the ones with more streaking failed

Continue on 9/4[edit]

Expected Sequence Expected Sequence Expected Sequence Expected Sequence Expected Sequence
M13R Orange Primer Sample Sequence Blue Primer M13F
CAGGAAACAGCTATGAC CCTTGCCAGCCCGCTCAG N/A CCTCCCTCGCGCCATCAG GTAAAACGACGGCCAG
Expected Reverse Sequence Expected Reverse Sequence Expected Reverse Sequence Expected Reverse Sequence Expected Reverse Sequence
M13R Orange Primer Sample Sequence Blue Primer M13F
GACTCGCCCGACCGTTCC N/A GACTACCGCGCTCCCTCC GACCGGCAGCAAAATG
Expected Reverse Complement Sequence Expected Reverse Complement Sequence Expected Reverse Complement Sequence Expected Reverse Complement Sequence Expected Reverse Complement Sequence
M13R Orange Primer Sample Sequence Blue Primer M13F
CTGAGCGGGCTGGCAAGG N/A CTGATGGCGCGAGGGAGG CTGGCCGTCGTTTTAC
Sequence 1 Sequence 1 Sequence 1 Sequence 1 Sequence 1
M13R Orange Primer Sample Sequence Blue Primer M13F
Sequence CTGAGCGGGC 363bp CCTCCCTCGCGCCATCAG N/A
Location 364-373 124-363 106-123 N/A
Number of A T C G
# 131 78 82 64
Sequence 2 Sequence 2 Sequence 2 Sequence 2 Sequence 2
M13R Orange Primer Sample Sequence Blue Primer M13F
Sequence CTGAGCGGGCTGGCAAGG 196bp CCTCCCTCGCGCCATCAG CTGGCCGTC
Location 304-321 107-303 89-106 633-641
Number of A T C G
# 67 59 30 41

Sequence 3 Sequence 3 Sequence 3 Sequence 3 Sequence 3 M13R Orange Primer Sample Sequence Blue Primer M13F Sequence CTGAGCGGGCTGGCAAGG 293bp CCTCCCTCGCGCCATCAG CTGGCCGTCGTTTTAC Location 294-311 124-293 106-123 405-420 Number of A T C G

  1. 102 68 80 41

Results & Improvements[edit]

  • Athurva and I both failed to generate bisulfite converted libraries. Could be due to human error while preparing the bisulfite conversion reagent (it was a new tube prepared by me), the protocol was simple and I am sure I didn't make mistake at that time, but I guess I will pay more attention when repeating the protocol.
  • Ordered new Imprint kit and we will test it out
  • Lyse cells with Naoh instead of NP40
  • Perform Etoh precipitation instead of column after MDA