Team:UC San Diego/Notebook

Protocols

protocol
Gene Fragment PCR

Reaction mixture: (20uL)

Master Mix (2X) 10ul
10uM Primer F 1ul
10uM Primer R 1ul
Water 7ul
Template fragment 1ul (~10ng)

PCR cycle:

Temp Time
Initiation 98 30sec
Cycles 30X 98
50 (or optimal annealing temperature
72		 5sec
30sec
20sec
Final 72 2min
Hold 4 forever
Gel Extraction
  1. Run gels to visualize PCR products
    1. Make 2% agarose gel with EB
    2. Load all (20ul) of PCR product mixed with appropriate amount of loading buffer
    3. Remember to use big combs, skip a lane between different samples if possible when load the gel: it will make gel-cutting easier
  2. Extract DNA from gels using Qiagen Gel Extraction kit
  3. Determine DNA concentration using nanodrop.
Gibson Assembly

Gene fragment pool preparation (5ul total)

Gibson1 (hMBD2):

Egfp-eh .06 uL
Hmbd2 0.5 uL
pET28a 3uL
water 0.5 uL
Master mix 2X 5ul

Gibson2 (mMBD1):

Egfp-em .5ul
Mmbd1 0.5ul
pET28a 3ul
water 0ul
Master mix 2X 5ul

Mixed and incubate at 50 with shaking for 1hr

Cell Transformation

Cell Transformation of Gibson Assembly Samples
Positive control of Gibson Assembly - Ampicillin Resistance
Competent cell: XL10 (50uL each)

    Transferred 1uL Gibson Assembly sample into competent cell, stored on ice for 40 min
  • Heat shock: 42 degree for 45 sec, put back onto ice
  • 450uL SOC medium into each
  • 37 degree incubation with shaking for ~1h
  • Spread on pre-made Amp or Kana (50ug/ml) plate, overnight (8:00PM to Next day 11:00AM, 15hr)
    Vector pACYC184
  • 37 shaker overnight
Mini Prep (Qiaprep)
  1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
  2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
  3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
  4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  5. Apply 800 µl of the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.
  6. Centrifuge for 30–60 s. Discard the flow-through.
  7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
  8. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
  9. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
  10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
Protein Culture

Starter culture:

  • Add 6mL autoclaved LB into a 50mL Falcon tube, with 6uL 50mg/mL Kanamycin stock
  • Pick a colony from the plate using a sterile pipet tip and put into the tube
  • Incubated with shaking at 37 degree, stopped when you see the LB became cloudy (usually around 3 hours)

Large culture:

  • Pre-heated the 2L flasks with LB at 37 degree, add 750uL 50mg/mL Kanamycin stock into each flask
  • After the flasks warmed up, add 1.5mL starter culture into each flask
  • Monitor the OD600 1h, 2h and more frequent when it goes above 0.2 (usually 2h30min-4h)
  • Cool the flasks on ice when OD600 is approaching 0.8
  • Take a gel sample with 100uL culture, centrifuged at full speed for 10min at 4 degree, discard the supernatant and resuspend the pellet in 6uL 300mM NaCl + 20mM Tris (pH = 8), add 6uL SDS-PAGE gel loading buffer
  • Add IPTG into each flask until 0.5mM and change the incubator to room temperature
  • Wait until the incubator has cooled, put the flasks in
    E.coli harvest:
  • A gel sample after induction: take 100uL culture, centrifuged at full speed for 5min, discard the supernatant, resuspend the pellet in 9uL 300mM NaCl + 20mM Tris (pH = 8), +3uL GLB
  • Clean two 250mL centrifuge bottles
  • Centrifuge the culture in centrifuge bottles at 8000rpm for 10min at 4 degree each time, discard the supernatant each time
    E.coli cell resuspension and lysis
  • Resuspend the pellet in ~25mL 300mM NaCl + 20mM Tris (pH = 8), remember to thoroughly pipet up and down or vortex to resuspend the pellet into a homogenous viscous solution, transfer the final solution into a 50mL Falcon tube
  • Add protease inhibitor and lysozyme to the resuspension, mix thoroughly by inverting the tube several times
  • Take enough liquid nitrogen to soak the tube in for ~15min
    DNAse
  • Thaw the lysate in room temperature water bath
  • Add DNAse with 75uL 1M MgCl2, 15uL 1M CaCl2, mix thoroughly by inverting the tube several times (should see lysate cluster together)
  • Put the lysate in room temperature water bath for ~1h, until less viscous (1000uL pipet can easily pipet up and down)
  • Take a gel sample of the lysate: 9uL lysate + 3uL GLB
  • Centrifuge the lysate at 10000rpm for 1h at 4 degree, separate the supernatant immediately after centrifuge
  • Take a gel sample from the supernatant: 9uL supernatant + 3uL GLB
  • Resuspend the pellet in ~5mL resuspension buffer, take a gel sample from the resuspension: 9uL resuspension + 3uL GLB
    Starter Culture of mMBD-eGFP, hMBD-eGFP
  • 6mL LB broth + 6uL 50mg/mL Kanamycin + colonies of mMBD-eGFP/hMBD-eGFP
  • Incubated at 37 degree with shaking
  • mMBD-eGFP starter culture completed in 3h: cloudy can barely see the toothpick
  • hMBD-eGFP starter culture remained clear: probably some problems with the plate --> redo BL21 transformation
    Large Culture of mMBD-eGFP (2*750mL)
  • Culture + Kanamycin to final conc. Of 50ug/mL, preheated at 37 degree
  • 1.5mL (1:500 dilution) starter culture added to each 750mL
  • Stop incubation after ~3h30min when OD600 = 0.74, cool on ice
  • A gel sample taken: 100uL medium centrifuged, pellet resuspended in 6uL resuspension buffer (300mM NaCl + 20mM Tris (8)), +6uL GLB
  • 89mg IPTG (final conc. 0.5mM) added to each 750mL
  • Incubated at 18 degree with shaking (8:15pm)
  • Induction kept for 16h
    Cell transformation of hMBD-eGFP (miniprep1)
  • An antibiotic free plate spread with 6uL 50mg/mL Kana diluted in 100uL solution, preheated at 37 degree
  • 1uL miniprep + 50uL BL21, stored on ice for 40min
  • Heat shock in 42 degree water bath for 45 sec, put back onto ice
  • Add 300uL SOC medium, incubated in 37 degree heatblock with shaking for 1h, put back onto ice
  • 100uL culture spread onto the Kana plate (7:45pm)
  • Results: incubated for ~16h, some colonies (10-20)
    Cell harvest & lysis of mMBD-eGFP
  • medium centrifuged in 2 250mL centrifuge bottle, each time 8000rpm, 10min, 4 degree
  • pellet resuspended in 25mL resuspension buffer (300mM NaCl + 20mM Tris (8))
  • ~10mg lysozyme added
  • froze in liquid nitrogen
    DNAse:
  • haw the cell lysate in water bath
  • 25uL DNAse, 75uL 1M MgCl2, 15uL 1M CaCl2 added to cell lysate
  • kept in water bath for 1h, less viscous
  • a gel sample taken from the lysate: 6uL + 6uL GLB
  • centrifuged in 2 25mL centrifuge bottle, 14500rpm, 1h, 4 degree
  • a gel sample taken from the supernatant: 6uL + 6uL GLB
    Ni column: new Ni column (3mL slurry)
  • column washed with water 3 times, resuspension buffer (300mM NaCl + 20mM Tris (8)) 3 times
  • DNAse supernatant loaded, a gel sample taken from the flow through (6uL + 6uL GLB)
  • column washed with resuspension buffer (300mM NaCl + 20mM Tris (8)) 3 times, a gel sample taken from the wash (6uL + 6uL GLB)
  • elution 1: 6mL 25mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
  • elution 2: 6mL 100mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
  • elution 3: 6mL 250mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
  • a gel sample taken from each of the elution (6uL + 6uL GLB)
DNA Hybridization
  • Get 1ml 3X SSC from 10X SSC (300ul + 700ul water)
  • Added 2ul for each complementary ssDNA and 46ul 3XSSC to 50ul
# Forward Reverse
1 0_0 0_0
2 0_0 M_0
3 M_0 M_0
4 0_M M_0
  • Hybridization setup: 98C for 5min, Ramp -1C/min for 70min, 4C Hold
  • After reaction, each tube should have 50ul * 4uM dsDNA = 0.2nmol DNA
    DNA-protein reaction
  • 50ul reaction 9ul dsDNA (0.036nmol) + (+0.5nmol) 4ul mMBD-eGFP + 37ul binding buffer
  • Pre-incubate mMBD-eGFP + binding buffer for 10min at RT
  • Incubate for 30min at RT
  • Binding samples: 10ul mixture with 2ul loading dye (loaded 10ul)
    DNA only samples
  • Dilute 9ul dsDNA in 41ulwater (labeled "1:5"), take 10ul mixed with 2ul loading dye (loaded 10ul)

Gel: 2% agarose in 1X TAE

EMSA:
Polyacrylamide Gel: (6%)
    3ml 30% PA (29:1) Bio-Rad
    3.75mL 2X TBE
    8.25mL DD water
    150ul 10%APS
    15ul TEMED
  • Wait 30min for gel to polymerize
  • Pre-run with 0.5X TBE at 4 degree
  • Gel 1 (wells not perfect): ladder, 2:1, 1:1, dsDNA, ssDNA (12uL each)
  • Gel 2: ladder, 3:1, 5:1, dsDNA (12uL each)
  • Run at 100V at 4 degree for ~1h
  • Stained in 50mL 0.5x TBE + 5uL SybrSafe for 30min
Protein-DNA interaction: (50uL each)
1:1 2:1 3:1 5:1
mMBD-eGFP 4 8 12 20
Binding buffer (5x) 10 10 10 10
H2O 27 23 19 11
dsDNA 9 9 9 9

5x binding buffer:
50mM HEPES (7.9)
15mM MgCl2
50% glycerol
5mM DTT
500mM KCl

5x gel loading buffer (no SDS):
50mM EDTA
16.5mM Tris-HCl (8)
Bromophenol blue

  • Pre-incubate protein + binding buffer + H2O at RT for 10min
  • + dsDNA, incubate at RT for 30min
  • +12.5uL 5x gel loading buffer (no SDS)
Graphene Oxide

GO assay target 5nM, 50ul reactions

    All in NEB buffer 2
  • Quenching assay
  • GO Concentration: 0.02mg/mL
  • DNA probe concentration: 50nM DNA-FAM
  • Incubation time: 10min
    Buffer (ph 7.9): NEB 2 (1X)
  • 50mM NaCl
  • 10mM Tris-HCL (8.0)
  • 10mM MgCl2
  • 1mM DTT
    Buffer (pH 7):
  • NEB 1 (should be available with Exo III)
  • 10mM Bis-Tris-Propane-HCl
  • 10mM MgCl2
  • 1mM DTT
    Prep:
  • Dilute GO in NEB 2 buffer (or 1, need comparison).
  • Dilute DNA probe stock in 10mM phosphate buffer pH 7.4
    Quenching (bold indicate main optimizable parameters)
  1. In black 96 well, add 50nM (final conc) DNA-FAM in at least three replicates (3+ negative control)
  2. To experimental groups. Add GO solutions with a final conc of 0.02mg/ml and extra water to a total of 50ul, tap to mix (or carefully pipette up and down), incubate for 10+ min
  3. At the same time add RNA-grade water to 50ul for all negative controls
  4. Read fluorescence with a excitation at 480nm, emission from 505 to 600nm
    Recovery
  1. Keep 1+ negative control, perform the 4 steps in Quenching assay
  2. Prepare serial target probe dilutions (final concentration: 0pM, 5pM, 10pM, 100pM, 500pm, 5nM, 50nM) If successful move forward to lower conc
  3. Add serial probe dilutions in phosphate buffer (or NEB 2?), mix, incubate for ? mins
  4. (need to confirm) if GO could be precipitate by centrifugation, centrifuge down, draw out supernatant and load to separate wells, resuspend with buffer (?), do it again
  1. In a 96 clear well plate add GO solution in three replicates
  2. Quenching assay: in
  3. add probe solution to wells, tap to mix, incubate for 10+ min
Protein-DNA interaction: (50uL each)
7:1 5:1
mMBD-eGFP 30 20
Binding buffer (5x) 10 10
H2O 1 11
dsDNA 9 9
Quenching Assay

DNA probe

    Required buffer: (self making or borrowing the stocks) pH is crucial
  • 10mL 10mM sodium phosphate pH 7.4 from 1M sodium phosphate
  • NEB II buffer (ask whether they have it, if not prepare a 5X stock)
    • 50mM NaCl
    10mM Tris-HCL (usually they have pH 8.0)
    10mM MgCl2
    1mM DTT
    pH 7.9
    Required equipment:
  • 96 well fluorescent plate, borrowed from Zhang's lab or the lab with Tecan, black/non black both are fine (transparent will be better if you can get one)
  • Tecan Reader
    Procedures:
  1. Get access to fluorescent labeled DNA order info from IDT: order number 15387329 (should have 3, they are in one of the envelopes on the desk)
  2. Follow instruction to dilute each DNA sample to 100uM with 10mM phosphate pH 7.4,
  3. Prepare 200ul 10pM, 1nM, 100nM for each fluorescent DNA with NEB II buffer. Take a bit 100uM and make serial dilutions with dilution factors not bigger than 10 (10uM, 1uM, 100nM…)
  4. Take 10ul 2mg/ml GO dispersion (in the right top drawer of our cleanest bench) and dilute to 0.02mg/ml in NEB II buffer by pipette mixing, invert tube couple times before use
  5. In 96 well plate, add 25ul fluorescent DNA dilutions in four replicates (see sample plate map). 0nM DNA = neat NEB II buffer. 250pM, 5nM follow same setup as 10pM (with+without GO)
  6. In "-with GO" wells, add 25ul 0.02mg/ml GO solution; In "-without GO" wells, add 25ul NEB II buffer
  7. Shake at Tecan reader and record fluorescence at 5min, 10min, 15min with excitation at 480nm, emission from 520nm
Slot Blot

Input: 100ng, about 6 pmol
2ul each ssDNA, 46 3X SSC (4uM)
load 2ul each slot (8pmol)

Procedures:
Each membrane: 12 slots, (2cm *2cm)
Condition: (Crosslink/not)* 0.05X, 0.005
X (4 total)

Soak blot paper with 6X SSC for 10min
Wash each slot with 0.4mL TE, wait for dry
Pipette 2ul to each slot, wait for dry
Wash each slot with 0.4ml 2X SSC, wait for dry

Crosslink or not
Soak membrane in PBS-T for 10min
Incubate in blocking buffer for 50min (2.5g milk in 50mL PBS-T
Dilute mMBD-eGFP in PBS-T (10mL for each), incubate overnight at 4

Wash membrane with PBS-T twice
Incubate with AntiHis-HRP

Notebook

We have also documented our overall lab activities through several notebooks.