TITLE: 3XFlag-pATn5 Protein Purification and MEDS-loading (5x scale, 2L volume)
AUTHORS: Terri Bryson, Steven Henikoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here we describe the method used to purify and load pATn5 for CUT&Tag, </span><a href="https://www.protocols.io/view/bench-top-cut-amp-tag-z6hf9b6/abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/bench-top-cut-amp-tag-z6hf9b6/abstract</span></a><span>.  This protocol was modeled after that in Picelli et al (Picelli et al., 2014) using the pTXB1-rbs_3XFlag-pATn5-FL plasmid available from Addgene: </span><a href="https://www.addgene.org/124601/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/124601/</span></a><span>.  It yields, as written, </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span> used at 1:200 dilution in CUT&Tag reactions, and </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span> for future use, both of which should be stored at -20C and can easily be scaled down. </span></div></div>

[STEPS]
?. [Day 1]
Streak E. coli glycerol stock containing pTXB1-rbs_3XFlag-pATn5-FL plasmid onto an LB-Amp100 plate, incubate  at
37 °C
?. [Day 2]
Select a single colony and make a patch plate for later, use within 1 week.
?. [Day 3: Culture and Induce]
Early morning, with a sterile toothpick swipe through a bacterial patch to inoculate  of LB broth+ carbenicillin 100ug/mL culture, let shake ~ .
30 ml
?. [Day 3: Culture and Induce]
Put  of LB+ carbenicillin 100ug/mL in 6L flask at   as well so it is already warm at the time of dilution.
1970 ml
37 °C
Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so it is potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, it is also more expensive.
?. [Day 2]
Make buffers and media.
See Materials section.
?. [Day 3: Culture and Induce]
After  incubation use all 30 ml of starter culture to inoculate larger vessel.
?. [Day 3: Culture and Induce]
Shake at , check OD600 periodically until it reaches O.D600 of 0.5-0.7. ~
37 °C
?. [Day 3: Culture and Induce]
Move to cold room to let cool ~
?. [Day 3: Culture and Induce]
Induce culture with addition of fresh 1M IPTG to 0.25mM final and incubate on the  shaker ~
18 °C
?. [Day 4: Pellet and Freeze]
Pellet bacteria by centrifugation: Divide the culture between 6x  bottles, centrifuge at 9500rcf, 4C for  in a GS3 Rotor, RC5C Sorvall centrifuge.
500 ml
?. [Day 4: Pellet and Freeze]
Discard the supernatant.
?. [Day 4: Pellet and Freeze]
Freeze bacterial pellets in dry ice and ethanol bath.
?. [Day 4: Pellet and Freeze]
Store at  freezer until ready to process.
-80 °C
?. [Day 5: Lyse, Chitin Binding]
Remove pellets from freezer, thaw on ice and resuspend together in a combined total of  of chilled HEGX Buffer with Roche Complete EDTA free protease inhibitor (PI) tablets. Keep on ice while working.
Need   today; make a total of 1L HEGX and add PI tablets as needed each day.
200 ml
?. [Day 5: Lyse, Chitin Binding]
Divide combined resuspension between 4x clean 100 ml plastic beakers nested in a larger plastic beaker filled with ice. See figure A.
?. [Day 5: Lyse, Chitin Binding]
While keeping in the ice nest, sonicate each sample 10-12 times,  each at 50% duty cycle and output level 7 on a Branson Sonifier 250 fitted with the Branson Ultrasonics™ Sonifier™ micro-tip. Between rounds of sonication keep in larger ice bucket (see figure A) and use a 2mL serological pipette to gently stir to facilitate cooling. Let each sample rest at least  between sonication rounds.
To prevent foaming keep the sonicator tip in the center of the sample. If excessive foaming occurs during sonication either wait for foam to settle or remove with a pipette and discard.
?. [Day 5: Lyse, Chitin Binding]
Transfer to fresh 50 mL conical tubes and pellet the lysate by centrifugation for , , at .
4 °C
Centrifuge: 16000 34
?. [Day 5: Lyse, Chitin Binding]
In  prepare 10x Econo-Pac® Chromatography Columns (BIO-RAD 732-1010), each holds  Set-up columns (we use a rack intended for Qiagen Maxi-preps, figure B). Gently invert Chitin Resin (NEB S6651S) to fully suspend and add 2.5mL to each column. Wash each with  of HEGX Buffer, drain immediately, don’t collect anything. Do not let resin dry out prior to addition of sample.
Chitin Binding: The remainder of the protocol is carried out at   or on ice. All buffers should be chilled on ice prior to use.
[room]
20 ml
20 ml
?. [Day 5: Lyse, Chitin Binding]
Add yellow cap to bottom of column.
?. [Day 5: Lyse, Chitin Binding]
Add supernatant from lysate spin to Chitin Resin slowly to prevent bubbles.
?. [Day 5: Lyse, Chitin Binding]
Cap column tightly and incubate  at  on nutator. Check for leakage after  and, wrap both ends in parafilm if necessary to prevent leakage.
4 °C
?. [Day 6: Wash and Cleavage Intein with DTT.]
Return columns to rack: remove yellow cap gently, hang rack over collection tube and uncap column. Drain unbound fraction into 50 mL tubes (save on ice or at  in the event troubleshooting is required later).
4 °C
?. [Day 6: Wash and Cleavage Intein with DTT.]
Wash column 2x with  HEGX collecting first wash into 50 mL tubes (save on ice or at  in the event troubleshooting is required later), the second wash can be wasted.
20 ml
4 °C
?. [Day 6: Wash and Cleavage Intein with DTT.]
Add yellow cap to bottom of column.
?. [Day 6: Wash and Cleavage Intein with DTT.]
Make  HEGX +PI with 100mM DTT and slowly add   to each sample.
65 ml
6 ml
?. [Day 6: Wash and Cleavage Intein with DTT.]
Cap column tightly and nutate in cold room ~
?. [Day 7: Elute & Dialyze]
Return columns to rack: remove yellow cap gently, hang in rack over collection tube and uncap column. Drain unbound fraction into 15 mL conical tubes, combine at end. Cover resin with additional  of buffer and save at  until purification is verified.
2 ml
4 °C
?. [Day 7: Elute & Dialyze]
Record elution volume. ~
50 ml
?. [Day 7: Elute & Dialyze]
Dialyze the protein as described in the following substeps.
?. [Day 8: Concentrate Protein]
Separate dialyzed protein between 4x Millipore Centrifugal Filter Units 30K, Amicon Ultra-15 Cat# UFC903024
?. [Day 8: Concentrate Protein]
Spin in Eppendorf 5804R at  and  for . Check at  intervals to assess progress, do not let volume drop below top of filters.
Centrifuge: 2880 34
4 °C
?. [Day 8: Concentrate Protein]
Recover concentrated sample from the upper filter portion of the tube, use an extra 500uL of buffer from lower catch tube to wash any extra protein off of each filter and add to collected protein.
Volume recovered from Concentrator: 7 mL + 2mL for rinsing filter = 9 mL
?. [Day 8: Concentrate Protein]
Add an equal volume of sterile 80% glycerol to the concentrated protein.  Combined with the glycerol from dialysis the final glycerol concentration is ~50%.
?. [Day 8: Concentrate Protein]
Store at .
-20 °C
?. [PAGE Analysis of pATn5 purification]
Run small volume of purified protein (and some from a previous batch if available) to compare yields. Include BSA standard range for quantification if desired. To the 3x Loading Dye from CSH Protocols, add 2-mercaptoethanol to 15% final before adding to the protein samples, heat to  for  cool back to  prior to loading onto gel.
Material: • Novex; 4-20% Tris-Glycine Gel 1.0 mm, 12 well WEDGE, Thermo Fisher Scientific cat# XP04120PK2 • Pierce™ Bovine Serum Albumin Standard Pre-Diluted Set, Thermo Fisher Scientific cat# 23208 • PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, Thermo Fisher Scientific cat 26620• 2-Mercaptoethanol, BIO-RAD cat#1610710 • 1xTris Glycine Buffer
100 °C
0 Room temperature
?. [PAGE Analysis of pATn5 purification]
To make the BSA Standards dilute the 250 ng/uL BSA to 125 ng/uL and use increasing volumes on the gel.
?. [PAGE Analysis of pATn5 purification]
Sample Prep and Gel Loading (figure D)  ABCD1Lanes L→RVolume of protein, uL3xLoading Dye            (+2-ME)1xTE2Protein Marker6--3125ng BSA13104250ng BSA2395500ng BSA4386750ng BSA63771.5ug BSA123-8Protein Marker6--9pATn5 -Batch 21.531010pATn5 -Batch 31.531011pATn5 -Batch 33.038
ABCD1Lanes L→RVolume of protein, uL3xLoading Dye            (+2-ME)1xTE2Protein Marker6--3125ng BSA13104250ng BSA2395500ng BSA4386750ng BSA63771.5ug BSA123-8Protein Marker6--9pATn5 -Batch 21.531010pATn5 -Batch 31.531011pATn5 -Batch 33.038
?. [PAGE Analysis of pATn5 purification]
After loading samples run gel at 30mA for ~ or until the loading dye leading edge runs to the bottom of the gel.
?. [PAGE Analysis of pATn5 purification]
Transfer gel carefully to covered dish with Coomassie stain gently shake at least  to  at .
0 Room temperature
?. [PAGE Analysis of pATn5 purification]
Replace stain with destain, add a rolled paper towel to dish to absorb excess Coomassie dye.
?. [PAGE Analysis of pATn5 purification]
Replace destain with water, scan gel for notes.
?. [Day 7: Elute & Dialyze]
Assemble 2 Slide-A-Lyzer 10K MWCO Dialysis CassettesUse a medium (1 ¼ inch) binder clip to secure a 2 inch stir bar and remove the clip handles gently. Use an 18-inch zip tie to secure a foam float to the cassette.  See figure C.Hydrate the cassette by submerging and use a binder clip to anchor the cassette at the proper depth by clipping the zip tie tail to the lip of the dialysis beaker. Let hydrate according to product instructions
?. [Day 7: Elute & Dialyze]
Using an 18 gauge needle load the Slide-A-Lyzer Cassettes with  each. Follow product literature on how to properly load cassettes.
25 ml
?. [Day 7: Elute & Dialyze]
In a 4 L beaker, add both cassettes and dialyze for - in  of Tn5 dialysis buffer with DTT added to 1.7mM just before use.
2.5 L
?. [Day 7: Elute & Dialyze]
Move samples to fresh Tn5 dialysis buffer, also  with DTT added to 1.7mM just before use, let dialyze  at .
2.5 L
4 °C
?. [Day 7: Elute & Dialyze]
Recover sample from cassette and combine into a fresh 50mL conical tube.  Hold on ice or at  until next steps. Overnight storage is ok if this falls into a weekend.
4 °C
Volume recovered from Dialysis: ~
?. [Annealing of MEDS Oligos]
ABCDEFGHIJKLMNOP1Tube #Seq NameSeq 5' to 3'ODVol. µlnmolµgLenMW% GC
ContentE260TmScalePurif.Vol in µl for 100 µMVol in µl for 200 µM21TN5MErev[Phos]CTGTCTCTTATACACATCT30dry178.81031.5195768.836.816778353.71.0 µmolSalt-Free178889432Tn5ME-ATCGTCGGCAGCGTCAGATGTGTATAAG
AGACAG161.5dry484.24959.63310242.851.533345670.91.0 µmolSalt-Free48421210.543Tn5ME-BGTCTCGTGGGCTCGGAGATGTGTATAA
GAGACAG188.3dry550.75839.5341060352.934195671.91.0 µmolSalt-Free55072753.5
ABCDEFGHIJKLMNOP1Tube #Seq NameSeq 5' to 3'ODVol. µlnmolµgLenMW% GC
ContentE260TmScalePurif.Vol in µl for 100 µMVol in µl for 200 µM21TN5MErev[Phos]CTGTCTCTTATACACATCT30dry178.81031.5195768.836.816778353.71.0 µmolSalt-Free178889432Tn5ME-ATCGTCGGCAGCGTCAGATGTGTATAAG
AGACAG161.5dry484.24959.63310242.851.533345670.91.0 µmolSalt-Free48421210.543Tn5ME-BGTCTCGTGGGCTCGGAGATGTGTATAA
GAGACAG188.3dry550.75839.5341060352.934195671.91.0 µmolSalt-Free55072753.5
?. [Annealing of MEDS Oligos]
Resuspend single-stranded salt-free oligos from Eurofins to 200uM in Annealing Buffer (10mM Tris , 50mM NaCl, 1mM EDTA).
?. [Annealing of MEDS Oligos]
Mix equal volumes of both complementary oligos (at equimolar concentration) in a  microfuge tube, mix well and aliquot to in smaller tubes appropriate for thermal cycler.  +  =  → 4 tubes @   +  =  → 4 tubes @
1.5 ml
[Tn5MErev]
[Tn5ME-A]
800 µl
200 µl
[Tn5MErev]
[Tn5ME-B]
800 µl
200 µl
?. [Annealing of MEDS Oligos]
In MJ Cycler PTC-200 use ANNEALOL program which starts with a  incubation and decreases temp in  increments incubating  each ending with a  incubation at  and holds at  afterwards. Store at .
95 °C
5 °C
25 °C
8 °C
-20 °C
?. [Annealing of MEDS Oligos]
Verify annealing:Run oligos, both ss and ds, on a 6% acrylamide 1xTBE (figure E) Lanes:1. 1KB+ Marker 3uL2. Rev+ ME-A 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE3. Rev+ ME-B 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE4. Rev 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE5. ME-A 0.5uL +1uL 6X Fermentas Orange Loading Dye +6uL TE6. ME-B 0.5uL +1uL 6X Fermentas Orange Loading Dye +6uL TE7. 100 bp Marker 3uL
While annealing efficiency is not 100% it is robust, excess ssDNA will not load onto transposase and will wash away during CUT&Tag protocol.
?. [Assembly of Tn5 with pre-annealed MEDS and in vitro assay to verify tagmentation activity]
With oligo-mer:The mixture is incubated for ~ at  and then stored at .
[100 uM Tn5ME-rev/A oligo]
[100 uM Tn5ME-rev/B oligo]
[3xFlag-pATn5 (in 50% glycerol)]
-20 Room temperature
-20 °C
?. [Testing transposase activity in vitro]
In vitro activity assay is performed on 150 ng HMW Lambda DNA (NEB# N3011). Unloaded pATn5 or loaded pATN5 without MgCl2 should have no impact on HMW DNA, loaded pATN5 in digestion buffer should digest HMW DNA to a smear.  See figure F.
?. [Testing transposase activity in vitro]
For each sample prepare the following reaction mix: 
 HMW DNA  5XTAPS-MgCl2-PEG8000 (water for no digestion buffer controls)  pATn5-MEDS complex
150 ng
4 µl
5 µl
?. [Testing transposase activity in vitro]
Incubate for  at .
55 °C
?. [Testing transposase activity in vitro]
To release and inactivate Tn5, add  of 1%SDS (disrupt protein DNA interaction).
2 µl
?. [Testing transposase activity in vitro]
Incubate for  at  or .
55 Room temperature
55 °C
?. [Testing transposase activity in vitro]
Add  proteinase K (20 mg/mL) to each reaction (degrade pATn5).
0.63 µl
?. [Testing transposase activity in vitro]
Incubate for  at .
55 °C
?. [Testing transposase activity in vitro]
Keep  or .
on ice
4 °C
?. [Testing transposase activity in vitro]
The samples can be analyzed by migration on 0.7% agarose gel in 1xTAE.
Gel Lanes: (figure F) 1. 150ng HMW lambda DNA 2. 1KB+ MW Marker 3. pATn5 (-) MEDS 4. pATn5 (+) MEDS, (-) digestion buffer 5. pATn5 (+) MEDS, (+) digestion buffer 6. pATn5[batch 2], (+) MEDS, (+) digestion buffer