TITLE: Generation and Purification of pTXB1.Tn5
AUTHORS: Ryan     Mulqueen, Andy Fields, Andrew    Adey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Generation of Tn5 transposome, protein purification and loading for the sci- family of protocols.</div><div class = "text-block">Protein purification of pTXB1.Tn5 transformed into T7 Express LysY (NEB C3031) cells; </div><div class = "text-block">reference is PTXB1-Tn5 Addgene #60240 from Picelli et al. Genome Res. 2014.''</div><div class = "text-block">This protocol is built largely off of work described in</div><div class = "text-block">Picelli, Simone, et al. "Tn5 transposase and tagmentation procedures for massively scaled sequencing projects." Genome research 24.12 (2014): 2033-2040. </div></div>

[STEPS]
?. [Prior to day 1]
Generate LB agarose plates with    following addgene suggested protocol. https://www.addgene.org/mol-bio-reference/#antibiotics
[Carbenicillin]
?. [Day 1: Grow fresh colonies]
Freshly streak out a LB agarose plates with    from the glycerol stock. Allow to grow overnight at  in an incubator.
[Carbenicillin]
30 °C
?. [Prior to day 1]
Streak out E. coli stab with pTXB1-Tn5 plasmid received from Addgene (https://www.addgene.org/60240/).
?. [Prior to day 1]
Grow out E. coli to generate long term storage stock in   in a cryovial tube as described in https://www.addgene.org/recipient-instructions/myplasmid/#long.Store at  indefinitely.
[glycerol]
-80 °C
?. [Day 2: Expand single colony]
Pick a single colony to inoculate   and incubate with shaking () at   overnight.
[LB/carb]
30 °C
?. [Day 3: Induce Expression]
Add 2mL inoculum (expanded liquid colony) to    in a 2 L capacity Erlenmeyer flask.
[LB/carb]
?. [Prior to day 1]
Select single colony with a sterile pipette tip and grow out colony in liquid LB with
[Carbenicillin]
?. [Prior to day 1]
Incubate with shaking () at   overnight.
30 °C
?. [Prior to day 1]
Extract plasmid using Qiagen Miniprep spin column following manufacturer's instructions.
?. [Prior to day 1]
Transform T7 Express LysY(NEB C3031) cells with extracted plasmid.
?. [Day 3: Induce Expression]
Grow to O.D.600 = .400-500 ( ) at   with shaking () , then let cool to  .
[roughly]
30 °C
0 Room temperature
?. [Day 3: Induce Expression]
Add IPTG to  final concentration (  of  stock) for induction at  -  on shaker with mild agitation ( ) overnight.
1 mL
18 °C
22 °C
We found no difference in temperature ranges from 18C to 22C during induction.
?. [Day 4: Cell Pelleting and Protein Extraction]
Spun down cells with a JA-10 rotor (Beckman Coulter) at   for   in 500 ml bottles (make sure bottles are balanced).
?. [Day 4: Cell Pelleting and Protein Extraction]
Decant supernatant and keep pellets on ice.
Safe stopping point: Pellets can be frozen in    and stored for a week.
?. [Day 4: Cell Pelleting and Protein Extraction]
Prepare  HEGX Buffer.  ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM20mL3NaClDry800mM46.8g4EDTA (pH 8.0)0.5M1mM2mL5Glycerol100%10%100mL6Triton-X100100%0.2%2mL
1 L
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM20mL3NaClDry800mM46.8g4EDTA (pH 8.0)0.5M1mM2mL5Glycerol100%10%100mL6Triton-X100100%0.2%2mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Perform steps 16 onwards in a cold room.Resuspend pellet in  HEGX Buffer and  of EDTA-free protease inhibitor tablets in 100 ml beaker. Keep everything at .
75 mL
[(ice chilled)]
3
4 °C
?. [Day 4: Cell Pelleting and Protein Extraction]
Sonicate for  total:Perform   pulses with a  break between each pulse. (30 pulses total).
[pulses]
Remove stir bar from beaker before sonicating. Keep everything on ice during  sonication. Avoid  foaming solution during sonication
?. [Day 4: Cell Pelleting and Protein Extraction]
Add a sterile magnetic stir bar and break up pellet on a  stir plate in a cold room.  Keep everything at .
4 °C
?. [Day 4: Cell Pelleting and Protein Extraction]
Spin down lysate in 2 x 50 mL oak ridge tubes in JA-16 or JA-20 rotor. Make sure tubes are balanced. for  ,
4 °C
?. [Day 4: Cell Pelleting and Protein Extraction]
Aliquot   supernatant for future protein gel to check induction and purity. Pour remaining supernatant into clean 100 ml beaker with stir bar.
30 µl
?. [Day 4: Cell Pelleting and Protein Extraction]
Precipitate DNA:To supernatant in beaker, add  dropwise of  Poly(ethyleneimine) Solution as it stirs on stir plate to precipitate DNA.    ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2Poly(ethyleneimine) Solution50%5%1mL3HEPES-KOH (pH 7.2)1M20mM200 uL
2 mL
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2Poly(ethyleneimine) Solution50%5%1mL3HEPES-KOH (pH 7.2)1M20mM200 uL
Add PEI solution very slowly, dropwise down sides of beaker by a transfer pipette.Solution will become cloudy. Do not precipitate DNA too quickly.
?. [Day 4: Cell Pelleting and Protein Extraction]
Spin down supernatant in 2 x 50mL oak ridge tubes in JA-16 or JA-20 rotor. Make sure tubes are balanced. for  ,
4 °C
?. [Day 4: Cell Pelleting and Protein Extraction]
Aliquot   supernatant for future protein gel to check induction and purity. Pour remaining supernatant (henceforth called Tn5 lysate) into clean 500 ml beaker on ice.Bring volume up to  ice cold HEGX Buffer.
30 µl
150 mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Chitin Column preparation and loading Tn5 lysate:Clamp column on a stand in cold room for gravity flow.
Column type: Kimble-Chase Flex-Column Economy Columns with two  3-way valve stop-cocksWe've been using a 30cm (1 cm ID) column which enables you to add 24 mls volume during elution.
?. [Day 4: Cell Pelleting and Protein Extraction]
Add of Chitin resin using 10 ml serological pipet to column and allow resin to settle and pack on bottom of column
10 mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Equilibrate resin with of HEGX by gravity flow. Discard flow through.
200 mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Load  of Tn5 Lysate by gravity flow over column. SAVE this flow through for future protein gel to check induction and purity.
150 mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Once all protein solution is loaded on column,  wash column with  HEGX by gravity flow. Discard any flow through.
200 mL
?. [Day 5: Elution and Dialysis]
Elution continued:Collect of flow through from column.
9 mL
?. [Day 5: Elution and Dialysis]
Dialyze against  of  Dialysis buffer for  to   in a cold room.4   ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM100mL3NaCl5M200mM40mL4EDTA (pH 8.0)0.5M0.2mM400uL5Glycerol100%20%200mL6Triton-X100100%0.2%2mL7Prior to adding DTT, Dialysis buffer can be stored at 4C8DTTDry2mM0.308g
1 L
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEPES-KOH (pH 7.2)1M100mM100mL3NaCl5M200mM40mL4EDTA (pH 8.0)0.5M0.2mM400uL5Glycerol100%20%200mL6Triton-X100100%0.2%2mL7Prior to adding DTT, Dialysis buffer can be stored at 4C8DTTDry2mM0.308g
We use a Slide-a-Lyzer G2 cassettes with 10 KDa cutoff (ThermoFisher, No. 87731).
?. [Day 5: Elution and Dialysis]
Exchange buffer with  of fresh dialysis buffer and dialyze overnight.
1 L
?. [Day 6: Size check and storage]
Measure O.D.280. It should be about 4.0.
?. [Day 6: Size check and storage]
Aliquot   supernatant for future protein gel to check induction and purity. Run an SDS-PAGE gel following manufacturer's instruction on aliquots from steps 20, 23, 27 and 34 to check purity, and induction.
30 µl
?. [Day 6: Size check and storage]
After dialysis, add approximately of sterile 100% glycerol to bring final concentration of glycerol to 55% (for approx. 15 ml  final volume).Aliquot in cryotubes and freeze at . Protein is good for at least 1 year with proper storage.
6 mL
-20 °C
?. [Day 4: Cell Pelleting and Protein Extraction]
Leave HEGX/DTT solution on column in cold room for  to
?. [Day 4: Cell Pelleting and Protein Extraction]
Then close stop-cock and cap stop-cock to prevent any liquid from leaking from column. Cap stopcock on top of column too.
?. [Day 4: Cell Pelleting and Protein Extraction]
Open bottom stopcock and let  of HEGX with  DTT to flow through column. Measure volume by allowing volume to flow into 15mL falcon tube.
11 mL
?. [Day 4: Cell Pelleting and Protein Extraction]
Add  of HEGX with  DTT directly to top of column material (Chitin) by a 25 ml serological pipette.  ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEGX Buffer1X1X24mL3DTTDry100mM0.37g
24 mL
ABCD1ReagentStock ConcentrationFinal ConcentrationAmount of Stock2HEGX Buffer1X1X24mL3DTTDry100mM0.37g
?. [Day 4: Cell Pelleting and Protein Extraction]
Elution:Allow all wash buffer to drain through column and close bottom stopcock.