Brandon:LabNotes/Project1/2014-10-21

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Tn5 production protocol and validations (10/21/2014 updated), Round 7[edit]

  • The last Tn5 isolation R6 was able to get better results then R2 with 8X dilution similar to nextera control. For R6 used a fresh transformation. Bradford assay results were bright blue.


  • This time will do fresh transformation again, leave at RT overnight for induction (hopefully A600 = 3.0), add 100 ug/ml ampicillin at beginning of IPTG induction, and before leaving at night. Will wash with wash buffer that is 37C.
    • hepes for washing, tris during dialysis


  • Maybe transposase has a inherent nuclease activity when unbound? will have to test nextera transposase again. Or there is nuclease contamination.



buffer https://www.neb.com/products/e6901-impact-kit Intein Mediated Purification with an Affinity Chitin-binding Tag

  • Grown on Luria broth, E. coli cells


  • Things in bold are changed or what was actually used and differed slightly from the protocol.


Supplies order list[edit]

Reagent Vender Catalog Number Unit Price
T7 Express lysY/Iq Competent E. coli (High Efficiency) NEB C3013I (6x0.2ml/tube) 140
Core Bio Service C3013I (6x0.2ml/tube) 113.16
NEB C3013H (20x0.05ml/tube) 179
Chitin Resin NEB S6651S (20ml) 69
Core Bio Service S6651S 57.93
NEB S6651L (100ml) 276
SOC Medium NEB B9020S (4x25ml medium) 71
Life/Invitrogen 15544-034 (10x10ml) 89.76
LB Agar Ampicillin - 50ug/ml, Plates Sigma-Aldrich L0168-10EA 32
LB Agar Plates with Ampicillin-100. 100mm Plates, 20 pack Core Bio Service L1004 24.5
Ampicillin Ready Made Solution, 100mg/ml, 0.2um Filtered Sigma-Aldrich A5354-10ML 79.1
Ampicilin, sodium salt
Agar (Purified) Sigma-Aldrich 05039-50G 19.4
LB Broth with agar (Lennox) Sigma-Aldrich L2897-250G 78.1
LB Broth (Lennox) Sigma-Aldrich L3022-250G 40.7
IPTG (powder, 1G) Thermo Scientific R0391 (1gram) 44
Sigma-Aldrich I6758-1G 56.6
Life/Invitrogen 15529-019 59.16
PEI Sigma-Aldrich P3143-100mL 38.5
Econo-Pac Chromatography Column Bio-Rad 732-1010 138
DTT Thermo Scientific R0861 (5grams) 78
Sigma-Aldrich D9779-5G 131
HEPES Solution (pH 7.0-7.6) Life/Gibco 15630-106 (20mL) 18.5
Triton X-100 (100mL) Sigma-Aldrich X100-100ML 32.9



Buffers List[edit]

USING ORIGINAL BUFFER SOLUTIONS FROM R1


  • 1X HEGX
20 mM Hepes pH 7.2
0.8 M NaCl
1 mM EDTA
10% glycerol
0.2% Triton-X 100


  • 2X HEGX to make 1L
40 mM Hepes pH 7.2   40 mL of 1M
1.6 M NaCl           93.405 g OR 320mL of 5M
2 mM EDTA            0.58448 g OR 4mL of 500 mM
20% glycerol         200 mL
0.4% Triton-X 100    4 mL


  • 2xTn5 dialysis Bf: WITH TRIS-HCL INSTEAD
                                1L (H20 added to vol.)
100 mM Tris HCl, pH 7.5         50 mL 2M OR 25 mL 4M
200 mM NaCl			11.69 g OR 40mL of 5M
0.2 mM EDTA			400 uL 500 mM
2 mM DTT			2 mL 1M (309 mg)
0.2% Triton X-100		2 mL Triton X-100
20% Glycerol			200 mL OR 252g 100% Glycerol


  • Tn5 storage Bf: WITH TRIS-HCL INSTEAD
                         100 mL (+42 mL H20)     1L                        2L
50 mM Tris HCl, pH 7.5   2.5 mL 2M               25 mL 2M OR 12.5 mL 4M    50 mL 2M
100 mM NaCl		 0.584g OR 2 mL 5M       5.844 g OR 20ml 5M        11.69 g OR 40ml 5M
0.1 mM EDTA		 20 uL 500 mM            200 uL of .5M            400 uL of .5M
1 mM DTT		 0.1 mL 1M               0.154 g                  0.309 g
0.1% Triton X-100       100 uL 100%             1 mL 100%                2 mL 100%
50% Glycerol		 50 mL OR 63g            500 mL                   1000 mL



Buffers etc to make checklist[edit]

  • start 5-10 mL cultures in morning
  • Autoclave LB, add amp, put into 37C overnight
  • make 2X HEGX
  • make PEI, .22 uM filter, pH to 7.5
  • make 2X Tn5 dialysis buffer
  • make tn5 storage buffer
  • make HEGX +100 mM DTT



I. Growth and Induction[edit]

  • 1. Transform C3013 E. coli cells (NEB) (C3013I/H, plasmid, SOC, AMP/LB broth/agar/culture plates) (use +/- controls of puc1 and no transformation)
    • a. For C3013H: Thaw a tube of T7 Express lysY/Iq Competent E. coli cells on ice for 10 minutes.
    • b. For C3013I: Thaw a tube of T7 Express lysY/Iq Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
    • c. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
    • d. Place the mixture on ice for 30 minutes. Do not mix.
    • e. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
    • f. Place on ice for 5 minutes. Do not mix.
    • g. Pipette 950 µl of room temperature SOC into the mixture.
    • h. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    • i. Warm selection plates to 37°C.
    • j. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. (use half plate for each, 25 uL, 25 uL of 1:10, 25 uL of 1:100)'
    • k. Spread 50-100 µl of each dilution onto a selection plate (100 ug/ml amp) and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or at 25°C for 48 hours.
  • 2. Pick colony for starter culture. Five cultures (~100 mL) of same clone grown up at 37C in 5-10ml overnight (O/N) (LB culture broth, amp at 100 µg/ml), starting in the morning. takes awhile to reach A600 of 0.9 the next morning. Put 1 liter LB into 37C incubator for next morning.
  • 3. In morning, 1 liter cultures inoculated with all starter cultures, 250 mL culture per 1L flask. 37C for 3hr to A600 ~ 0.9. (1L LB culture broth, amp at 100 µg/ml, 100 mg per 1L) (spectrophotometer to measure A600 in cuvettes)
  • 4. Chill cultures in ice water bath to about 10C CHILLED TO 23C, ADDED IPTG, THEN SHOOK AT RT
  • 5. Add 1M IPTG to 0.25mM (IPTG) (250 uL of 1 M IPTG per 1 L culture?, 4000X dilution)
  • 6. Grow 23C for 4hr to A600 ~ 3.0, or 15C overnight A600 ~ 1.175
  • 7. Centrifuge 6000xg for 15 mins. Dump supernatent and Freeze pellet -80C overnight in the centrifuge bottles. Thaw in 80ml HEGX next morning.
  • STOPPING POINT

II. Sonicate (mixonix sonicator 3000)[edit]

  • 1. Make HEGX (HEGX: Tris-HCl (pH 7.5), NaCl, EDTA, Glycerol, TX-100, Complete)
  • 2. Resuspend pellet in 80 mL HEGX @ 0-4C, with complete protease inhibitors. added 8 roche complete protease inhibitor tablets (1 per 10 mL)
  • 3. Sonicate in HEGX in steel container 10-12 cycles of 45-50 bursts (0.5 seconds ON, 0.5 seconds OFF, (50% duty cycle)), output power level 7.0. ~15 minutes sonicating total. Temp should not go over 10C, keep consistently on salt ice bath at <4C, or cool on salt bath to -2C to -3C in between cycles. Clean in EtOH/MilliQ water and then immerse 1.5X tip diameter into solution, use 1/2 inch tip. Monitor temp with temperature probe with instantaneous feedback. Check transparency to see cell lysis after every few cycles. protein precipitates with over sonication. Solution turns translucent and less murky.
  • 4. Pellet 15K rpm, 30min 4C (BIG OL’ CENTRIFUGE)
  • 5. Keep supernatant and discard pellet


III. PEI precipitation (Mag stirrer)[edit]

  • 1. Make PEI:
    • a. total 12.5 mL
    • b. 2.7g 50% PEI
    • c. 5 ml H2O
    • d. 1.5 ml 37% HCl
    • e. 2 mL 5 M NaCl
    • f. 125 uL 2M Tris HCl pH 7.5
    • g. 7.5 uL 0.5 M EDTA
    • h. 250 uL Triton X-100 (0.2% TX100)
    • i. Adjust to 12.5 mL, 10% PEI (check pH and bring to pH 7.5)
    • j. Filter solution with .22 uM filter
  • 2. Add dropwise 2.1ml 10% PEI, stir briefly (PEI Sigma P3143)
  • 3. Collect precipitate at 12K, 10min, 4C; KEEP supernatant


IV. Chitin column[edit]

Without transposons


  • 1. Wash Chitin resin at RT with RT HEGX. take 17.5 mL of resin, let settle or spin down in 50 mL conical at 100xg for <1 minute with slow setting 5 for deceleration of centrifuge. Wash 5X with 1X HEGX, remove supernatent with aspirator.
  • 2. Add supernatant from PEI to washed chitin resin in conical. Lightly mix on shaker for ~3.5 hours at 4C
  • 3. Wash bound chitin beads 2X with RT 1X HEGX at RT in 50 mL conical. spin down at 100 xg for <1 minute with setting of 5 for deceleration of centrifuge, remove supernatent with aspirator.
  • 4. Add solution from conical to chromatography column, after draining put upper bed support on top, and wet with buffer.
  • 5. Wash with 20-30 volumes (200-300 ml) HEGX at gravity flow (2-4 ml/min) at RT OR wash overnight at 0.2ml/min at 4C
  • 6. Add 30+ ml Cleavage buffer +100 mM DTT to column.
    • a. 694 mg DTT in 45 mL
    • b. 22.5 mL 2X HEGX
    • c. 21.8 mL H20
    • d. 45 mL Final volume
  • 7. Run out until ~7-8 mL (not including beads) cleavage buffer is in column, stop flow and leave at 4C for at least 36 hours (over weekend) Did for 63 hours last time
  • 8. Elute first fraction of ~5 drops to discard (dead volume, no protein). Then elute ~10 fractions of 10 drops (~500 uL), then 12x20 (~1 mL) drop fractions.
  • 9. (optional) Leave the column in the remaining Cleavage buffer +100 mM DTT for 24h. (Checked: Up to 30% may be in elute2).

With Transposons

  • 1. Add supernatant from PEI precipitation to 10ml Chitin column at 0.4ml/min in HEGX (~3.5 hours) at 4C.
  • 2. Wash with 10-20 volumes HEGX, (100-200 mL) at gravity flow at 4C (~4 mL a minute = 25 mins)
  • 3. Repeat wash with 2 column volumes (200 mL) degassed HEGX at gravity flow at 4C
  • 4. Add 8 mL degassed HEGX containing (at least) 200 nmoles of mixed Tn5MEDS-A/-B oligonucleotides to the column (oligos, degassed? HEGX)
  • 5. Leave column at room temp overnight
  • 6. Wash with 20 volumes HEGX to remove free transposons at 4C.
  • 7. Add 25 ml HEGX, 100 mM DTT to column.
    • a. 385.6 mg DTT in 25 mL (2.5 mL 1M DTT)
    • b. 12.5 mL 2X HEGX
    • c. 12.1 mL H20
    • d. 25 mL Final volume
  • 8. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend)
  • 9. Elute 6 fractions of 20 drops, then 12x30 drop fractions.
  • 10. (optional) Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).


V. Dialysis (dialyzer)[edit]

  • 1. Make 2X dialysis buffer and chill at 4C. Pool fractions with highest concentration. Test with biorad protein assay (bradford asssy) using 25 uL and 1 uL sample. pooled fractions 1-6, 7-10
  • 2. Dialyze at 4C versus one change of 1 L 2x Tn5 dialysis buffer. Dialysis is for 2 hours with intermittent stirring every ~30 mins. (will concentrate sample some)
  • 3. Measure A280 with nanodrop to get concentration (can take a small amount our of dialysis bag and leave the rest in, then don't have to change dialysis bags)
  • 4. Dialyze at 4C with 2 changes of 1 Liter tn5 storage buffer (50% glycerol). Dialysis is for 2 hours with intermittent stirring every ~30 mins. (will concentrate sample ~2X)


  • 2xTn5 dialysis Bf: WITH TRIS-HCL INSTEAD
                                1L (H20 added to vol.)
100 mM Tris HCl, pH 7.5         50 mL 2M	
200 mM NaCl			11.69 g OR 40mL of 5M
0.2 mM EDTA			400 uL 500 mM
2 mM DTT			2 mL 1M (309 mg)
0.2% Triton X-100		2 mL Triton X-100
20% Glycerol			200 mL OR 252g 100% Glycerol


  • Tn5 storage Bf: WITH TRIS-HCL INSTEAD
                         100 mL (+42 mL H20)      1L                  2L
50 mM Tris HCl, pH 7.5   2.5 mL 2M                25 mL 2M            50 mL 2M
100 mM NaCl		 0.584g OR 2 mL 5M        5.844 g OR 20ml 5M  11.69 g OR 40ml 5M
0.1 mM EDTA		 20 uL 500 mM             200 uL of .5M       400 uL of .5M
1 mM DTT		 0.1 mL 1M                0.154 g             0.309 g
0.1% Triton X-100       100 uL 100%              1 mL 100%           2 mL 100%
50% Glycerol		 50 mL OR 63g             500 mL              1000 mL


  • 5. Should be concentrated now, and in storage buffer (50% glycerol). Amicon filters results in protein denaturation and thus avoid using. Store at -20C.

If not dialyzing with storage buffer

  • 1. After dialysis if protein is concentrated enough an be used directly for transposome complex assembly. If not then put into storage buffer by adding 1.05 volume 99.5% glycerol and 0.33 volume of 2x Tn5 dialysis buffer (final 55% glycerol) and store at -20C.
  • 2. (optional) If not concentrated enough, use Amicon Ultracel 30 filters and spin at 3000 rcf for 25 mins with mixing every 10 mins at 4C until final OD280 >3.0. Then can perform the above step of transposome complex assembly and preparation for storage at -20C. concentrating with filters greatly reduces specific activity of Tn5



Protocol summary from Gosta[edit]

Lab protocol for Tn5 prep 2014-03-27 Gösta Winberg and Rickard Sandberg added by us:

  • Tues: Transform C3013 bacterias and grow up plate overnight (amp selection)
  • Wed: Inoculate (several, like five 5 mL cultures with same clone in the morning. A600 of .9 takes a long time to reach) 5 mL culture and grow up overnight at 30C (amp selection).
  • Thurs: add all cultures to 1L culture, grow up, express protein, centrifuge, freeze -80 O/N
  • Fri: sonicate pellet debris, PEI precip, chitin column binding/wash, DTT cleavage and leave over weekend at 4C.
  • Mon: dialysis with 2X dialysis buffer, A280 molarity calculation. dialysis with storage buffer, and tn5 fragmentation assay


Thursday 27/3:

  • Thaw cell pellet and sonic in 80 mL HEGX, pellet debris.
  • PEI precipitation, pellet PEI-DNA precipitate.
  • Supernate to 7 mL Chitin column at 0.4 mL/min = 3,5 h.
  • Wash HEGX, 100 mL at 4 mL/min =gravity flow, 25 min
  • Last wash degassed 2 col. vol.
  • Add 8 mL degassed HEGX containing 200 nmoles of MEDS oligo (mixed 1030+1031 ds oligos). (Better to use at least 2x molar excess MEDS oligo)
  • A 7 mL column can bind max 200 nmoles of Tn5 fusion protein (28 nmoles/mL).
  • Leave O/N at RT for binding to the Tn5 protein.


Friday 28/3:

  • Move column to +4. Wash with HEGX to remove excess unbound oligo.
  • Add 20 mL HEGX, 100 mM DTT. Drain out 15 mL , then close the valve.
  • Leave column at +4 over weekend.


Mo 31/3:

  • Elute 4 fractions of 20 drops, then 12 x 40 drop fractions.
  • Test with BioRad protein assay: 25 uL + 1 uL of column fraction.
  • Pool most active fractions (most intense blue color).
  • Dialyze vs 2 x 1L Tn5 dialysis buffer at 4 C.


Tue 1/4:

  • Read OD280 vs dialysis buffer as blank.
  • Calculate Tn5 molarity (only approximately since the oligo influences the readings. Run Tn5 assay with 50 ng *High MW DNA as substrate, Run 1% agarose gel to check activity.



Validation of results[edit]

  • f1 0.45 average abs; 5.202 uM, 3.0 mL total, final of 1.2 mL, 42.86% concentration, 7.43 uM
  • f2 0.64 average abs; 7.398 uM, 3.4 mL total, final of 1.4 mL, 41.66% concentration, 10.48 uM
  • calculation for uM. 100 uM/8.65 = X uM/abs


10/28/2014

  • incubation with transposase only to test nuclease contamination
  • with 6 ng DNA
  • incubated reaction at 55C for 5 mins, followed by qiagen protease digestion, 50C for 10 mins, 70C for 20 mins.
  • reaction composition
1   uL pure Jurkat DNA (6 ng)
2-3 uL H2O
1-2 uL transposase (diluted if stated)
__________
5 uL total
samples.
1. R7f1, 2 uL tsase, 6 ng DNA
2. R7f1, undil. tsase, 6 ng DNA
3. R7f1, 2X dil. tsase, 6 ng DNA
4. R7f1, 4X dil. tsase, 6 ng DNA
5. R7f1, 8X dil. tsase, 6 ng DNA
6. R7f2, 2 uL tsase, 6 ng DNA
7. R7f2, undil. tsase, 6 ng DNA
8. R7f2, 2X dil. tsase, 6 ng DNA
9. R7f2, 4X dil. tsase, 6 ng DNA
10. R7f2, 8X dil. tsasa, 6 ng DNA
11. nextera, 6 ng pure DNA


  • still has nuclease activity. given washing conditions, would lean more toward tn5 having a inherent nuclease activity.

File:ZhangLab 2 2014-10-28 12hr 32min-labeled.jpg

IVT Protocol[edit]

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of t7tspn-top2 transposome

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of t7tspn-top2(8/22) and T7tspn-NoP-bot(8/22).
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
0.5 uL of annealed transposon
0.5 uL of 100% sterile glycerol
1.0 uL of Ez-TN5 transposase
  • store at -20, is good for a year


Generation of custom barcode (3' side of fragments)illuminia adaptor transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (sc1-T7tspn-NoP-bot, "pMENTS") and (sc-ILA-adaptor).
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed Nextera transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer


CELL WASHING - (wash cells with PBS)

  • 1. count cells, spin down all cells at 250-500 g for 5 minutes
  • 2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XXXX cells/uL
  • 3. Aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute LB to 2X, or as needed (from 10X stock)
  • 2. refrigerate or warm LB or keep at RT
  • 3. add lysis buffer to cell aliquots, mix briefly (mineral oil optional)
  • 4. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS

2003) in nuclease free H2O.



4. transposition reaction

  • add the below into one tube and incubate at 37C FOR 30 mins.
1.0 uL washed cells
1.0 uL 2X Lysis Buffer
1.0 uL 5X custom tagmentation buffer (buffer D6)
1.0 uL prepared transposomes
1.0 uL Nuclease free water
___________
5.0 uL total solution


5. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O)
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 1 uL 5X taq polymerase, run at 72C for 3 minutes. (OR 6 uL taq2X) (same as nextera)


7. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  bring to 20 uL with Nuclease free water
  X   uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  1   uL   10 mM ATP
  1   uL   10 mM CTP
  1   uL   10 mM GTP
  1   uL   10 mM UTP
  2   uL   T7 Enzyme Mix


b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)



8. Clean with Zymo clean and concentrator now

  • elute samples in 10 uL of N-H2O
  • quanitate with Qubit or on TBU gel.


AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


Results


  • TBU after IVT
  • seems like 4-8X diluions on R7f2 are still best. extra washing with 37C wash buffer didn't make much difference. probably has to do with buffers for efficiency now.
samples.
1. R7 f1, undil tsase, 6 ng DNA
2. R7 f1, 2X dil. tsase, 6 ng DNA
3. R7 f1, 4X dil. tsase, 6 ng DNA
4. R7 f1, 8X dil. tsase, 6 ng DNA
5. R7 f1, 16X dil. tsase, 6 ng DNA
6. R7 f2, undil tsase, 6 ng DNA
7. R7 f2, 2X dil. tsase, 6 ng DNA
8. R7 f2, 4X dil. tsase, 6 ng DNA
9. R7 f2, 8X dil. tsase, 6 ng DNA
10. R7 f2, 16X dil. tsasa, 6 ng DNA


File:ZhangLab 2 2014-10-31 13hr 27min-labeled.jpg


  • Example of Tn5 band from gosta

File:ZhangLab 2 2014-06-11 Gosta SDS-PAGE gel image.png