Brandon:Protocols/tn5 production
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Tn5 production protocol and validations[edit]
- Order now: C3013, SOC, AMP culture plates, AMP, IPTG, HEGX, PEI, chitin resin (http://www.neb.com/~/media/Catalog/All-*Products/BA2F358333E84BE7B55B64ABE796DC57/Datacards%20or%20Manuals/S6651Datasheet-Lot0121203.pdf)/columns (http://www.bio-rad.com/en-us/sku/732-1010-econo-pac-chromatography-columns), DTT, Tn5
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 actuaally 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]
- 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 2 mM EDTA 0.58448 g or 4 mL of 500 mM 20% glycerol 200 mL 0.4% Triton-X 100 4 mL
- 2xTn5 dialysis Bf:
1L (H20 added to vol.) 100 mM Hepes, pH 7.2 100 mL 1M or 23,83 g 200 mM NaCl 11.69 g NaCl 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 252 g 100% Glycerol
- 2xTn5 dialysis Bf: WITH TRIS-HCL INSTEAD
1L (H20 added to vol.) 100 mM Tris HCl, pH 7.5 100 mL 1M 200 mM NaCl 11.69 g NaCl 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 252 g 100% Glycerol
- Tn5 storage Bf:
100 mL (+42 mL H20) 1L 2L 50 mM Hepes, pH 7.2 5 mL 1M 11.915 g 23.83 g 100 mM NaCl 2 mL 5M 5.844 g 11.69 g 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 1 mL 10% (100 uL 100%) 1 mL 100% 2 mL 100% 50% Glycerol 63 g 100% Glycerol 500 mL 1000 mL
- Tn5 storage Bf: WITH TRIS-HCL INSTEAD
100 mL (+42 mL H20) 1L 2L 50 mM Tris HCl, pH 7.5 5 mL 1M 50 mL 1M 100 mL 1M 100 mM NaCl 2 mL 5M 5.844 g 11.69 g 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 1 mL 10% (100 uL 100%) 1 mL 100% 2 mL 100% 50% Glycerol 63 g 100% Glycerol 500 mL 1000 mL
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, 200 uL, 50 uL, 50 uL of 1:10, 50 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 of same clone grown up at 30C in 5ml 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. 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
- 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
- 7. Centrifuge and freeze pellet (if resuspending to isolate pellet do in HEGX) -70C overnight and thaw in 80ml HEGX (BIG OL’ CENTRIFUGE,Scupula, Culture tubes 6000xg for 8 minutes)
- STOPPING POINT
II. Sonicate (mixonix sonicator 3000)[edit]
- 1. Make HEGX (HEGX: Hepes-KOH (pH 7.2), 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)). ~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. 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. 0.25 mL 1 M Hepes pH 7.2
- g. 7.5 uL 0.5 M EDTA
- h. 0.25 mL 10% Triton X-100
- 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. Prepare chiten columns. pour 12 mL of chitin resin into column, after draining put upper bed support on top, and wash with 5 column volumes of buffer. use stopcock. Now ready for use
- a. OR take 12 mL of resin, let settle or spin down in 50 mL conical at 200xg for <1 minute., remove supernatant, and wash with 4 volumes column buffer, removing supernatent with aspirator. Repeat wash 5X.
- b. Add supernatant from PEI to washed chitin resin in conical. Lightly mix on shaker for 3.5 hours at 4C.
- c. Add solution from conical to chromatography column, after draining put upper bed support on top, and wet with buffer.
- d. proceed to step 3.
- 2. Add supernatant from PEI precipitation to 10ml Chitin column at 0.4ml/min in HEGX (~3.5 hours) (Chitin Resin, column?,lots of HEGX)
- 3. Wash with 20-30 volumes (200-300 ml) HEGX at gravity flow (2-4 ml/min) at 4C OR wash overnight at 0.2ml/min at 4C
- 4. 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
- 5. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend) did for 63 hours
- 6. Elute 6 fractions of 20 drops, then 12x30 drop fractions.
- 7. (optional) Leave the column in the remaining HEGX, 1M 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)
Tn5 dialysis buffer:
100mM Hepes, pH 7.2 0.2M NaCl 0.2mM EDTA 2mM DTT 0.2% TX100 20% Glycerol
Tn5 storage buffer:
50 mM Hepes, pH 7.2 100 mM NaCl 0.1 mM EDTA 1 mM DTT 0.1% Triton X-100 50% Glycerol
- 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]
- Successfully completely fragmented 6 ng or DNA, and partially fragmented 25 ng DNA. SDS-PAGE gel matches that which was provided by Gosta and Rickard.
- After concentrating in amicon filters, A600 was 7.44 or 86.01 uM. Thus final concentration after added 1.05 volumes glycerol, .33 volumes 2X tn5 dialysis buffer (total of 2.38X dilution factor), is 36.13 uM in 5.84 mL.
6/10/2014
- with 25 ng Jurkat DNA (unconcentrated transposase)
- annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30)
- tsp complex made by incubating at RT for ~30 mins
- reaction composition
2 uL 5X custom tagmentation buffer (D6) 1 uL pure Jurkat DNA (25 ng) 1 uL tspsome 1 uL 2X Lysis Buffer 5 uL Nuclease free water _____________ 10 uL total
File:ZhangLab 2 2014-06-10 20hr 02min-labeled.jpg
6/11/2014
- with 6 ng DNA (unconcentrated transposase)
- annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30)
- tsp complex made by incubating at RT for ~30 mins
- reaction composition
1 uL 5X tagmetnation buffer 1 uL pure Jurkat DNA (6 ng) 1 or 2 uL transposome complex 1 uL 2X Lysis buffer 1 or 0 uL H2O
File:ZhangLab 2 2014-06-11 12hr 59min-labeled.jpg
- Example of Tn5 band from gosta
File:ZhangLab 2 2014-06-11 Gosta SDS-PAGE gel image.png
- original SDS-PAGE gel image
File:ZhangLab 2 2014-06-11 SDS-PAGE gel tn5 original image.jpg
- SDS-PAGE gel with ladder labeled