Brandon:Protocols/tn5 production: Difference between revisions

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>Bsos
(Created page with "==Tn5 production protocol and validations== *Order now: C3013, SOC, AMP culture plates, AMP, IPTG, HEGX, PEI, chitin resin (http://www.neb.com/~/media/Catalog/All-*Products/B...")
 
>Bsos
 
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  0.2% Triton-X 100
  0.2% Triton-X 100


*2X HEGX              to make 1L
*2X HEGX               
                      to make 1L
  40 mM Hepes pH 7.2  40 mL of 1M
  40 mM Hepes pH 7.2  40 mL of 1M
  1.6 M NaCl          93.405 g
  1.6 M NaCl          93.405 g
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*2xTn5 dialysis Bf:  1L (H20 added to vol.)
*2xTn5 dialysis Bf: 
                                1L (H20 added to vol.)
  100 mM Hepes, pH 7.2 100 mL 1M or 23,83 g
  100 mM Hepes, pH 7.2 100 mL 1M or 23,83 g
  200 mM NaCl 11.69 g NaCl
  200 mM NaCl 11.69 g NaCl
Line 95: Line 97:
  20% Glycerol 252 g 100% Glycerol
  20% Glycerol 252 g 100% Glycerol


*Tn5 storage Bf: 100 mL: ( Mix + H2O, 42 mL)
50 mM Hepes, pH 7.2 5 mL 1M
100 mM NaCl 2 mL 5 M
0.1 mM EDTA 20 uL 500 mM
1 mM DTT 0.1 mL 1M
0.1% Triton X-100 1 mL 10%
50% Glycerol 63 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




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**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)''''
**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.
**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. 1L cultures inoculated with ( colony grown up in 5ml O/N, inoculate 1L in morning) (1L LB culture broth, amp at 100 μg/ml, 100 mg per 1L)
*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. 37C for 3hr to A600 ~ 0.9 (nanodrop to measure A600)
*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. Add 1M IPTG to 0.25mM (IPTG)  (250 uL of 1 M IPTG per 1 L culture?, 4000X dilution)
*4. Chill cultures in ice water bath to about 10C
*5. Chill cultures in cold water 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
*6. Grow 23C for 4hr to A600 ~ 3.0, or 15C overnight
*7. Centrifuge and freeze pellet -70C overnight and thaw in 80ml HEGX (BIG OL’ CENTRIFUGE,Scupula, Culture tubes 6000xg for 15 minutes)
*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
*STOPPING POINT


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===II.  Sonicate (mixonix sonicator 3000)===  
===II.  Sonicate (mixonix sonicator 3000)===  
*1. Make HEGX (HEGX: Hepes-KOH (pH 7.2), NaCl, EDTA, Glycerol, TX-100, Complete)
*1. Make HEGX (HEGX: Hepes-KOH (pH 7.2), NaCl, EDTA, Glycerol, TX-100, Complete)
*2. Resuspend pellet in 80 mL HEGX @ 0-4C, in a ultracentrifuge bottle, with complete protease inhibitors. '''added 8 roche complete protease inhibitor tablets (1 per 10 mL)'''
*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 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.
*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)
*4. Pellet 15K rpm, 30min 4C (BIG OL’ CENTRIFUGE)
*5. Keep supernatant and discard pellet
*5. Keep supernatant and discard pellet
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**g. 7.5 uL 0.5 M EDTA
**g. 7.5 uL 0.5 M EDTA
**h. 0.25 mL 10% Triton X-100
**h. 0.25 mL 10% Triton X-100
**i. Adjust to 12.5 mL, 10% PEI (check pH and adjust as needed). Bring to pH 7.5
**i. Adjust to 12.5 mL, 10% PEI (check pH and bring to pH 7.5)
**j.    Filter solution with .22 uM filter
**j.    Filter solution with .22 uM filter
*2. Add dropwise 2.1ml 10% PEI, stir briefly (PEI Sigma P3143)
*2. Add dropwise 2.1ml 10% PEI, stir briefly (PEI Sigma P3143)
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'''Without transposons'''
'''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. Now ready for use
*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. Repeat wash 5X.
**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 and let sit for 3.5 hours.
**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.
**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.
**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) '''Incubated for 1 hr 45 mins'''
*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) OR wash overnight at 0.2ml/min
*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 20 ml HEGX, 100 mM DTT to column.
*4. Add 25 ml HEGX, 100 mM DTT to column.
** . 308 mg DTT in 22 mL
**a. 385.6 mg DTT in 25 mL (2.5 mL 1M DTT)
**a. 10 mL 2X HEGX
**b. 12.5 mL 2X HEGX
**b. 2.0 mL 1M DTT '''added 309 mg DTT to solution'''
**c. 12.1 mL H20
**c. 8.0 mL H20
**d. 25 mL Final volume
**d. 20 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'''
*5. Run out 5 mL, stop flow and leave at 4C for at least 36 hours (over weekend) ''' did for 67 hours, 30 mins'''
*6. Elute 6 fractions of 20 drops, then 12x30 drop fractions.
*6. Elute 4 fractions of 20 drops, then 12x40 drop fractions. Test concentration (nanodrop)
*7. (optional) Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).  
*7. Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).  
 
'''With Transposons'''
'''With Transposons'''
*1. Add supernatant from PEI precipitation to 10ml Chitin column at 0.4ml/min in HEGX (~3.5 hours) (Chitin Resin, column?,lots of HEGX)
*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 (~4 mL a minute = 25 mins)
*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
*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)
*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
*5. Leave column at room temp overnight
*6. Wash with 20 volumes HEGX to remove free transposons
*6. Wash with 20 volumes HEGX to remove free transposons at 4C.
*7. Add 20 ml HEGX, 100 mM DTT to column.
*7. Add 25 ml HEGX, 100 mM DTT to column.
**a. 308 mg DTT in 22 mL
**a. 385.6 mg DTT in 25 mL (2.5 mL 1M DTT)
**b. 10 mL 2X HEGX
**b. 12.5 mL 2X HEGX
**c. 2.0 mL 1M DTT
**c. 12.1 mL H20
**d. 8.0 mL H20
**d. 25 mL Final volume
**e. 20 mL Final volume
*8. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend)
*8. Run out 15 mL, stop flow and leave at 4C for at least 36 hours (over weekend)
*9. Elute 4 fractions of 20 drops, then 12x40 drop fractions. Test concentration (nanodrop)
*9. Elute 6 fractions of 20 drops, then 12x30 drop fractions.
*10. Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).  
*10. (optional) Leave the column in the remaining HEGX, 1M DTT for 24h. (Checked: Up to 30% may be in elute2).
 




===V. Dialysis (dialyzer)===
===V. Dialysis (dialyzer)===
*1. Pool fractions with highest concentration '''pooled fractions 1-5, 6-8'''
*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 versus two changes of 1 L 2x Tn5 dialysis buffer at 4C (Tn5 dialysis buffer: 100mM Hepes, pH 7.2, 0.2M NaCl, ,0.2mM EDTA, 2mM DTT, 0.2% TX100, 20% Glycerol)
*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. Determine if need to concentrate (if OD280 >3.0, the dialyzed Tn5 can be used directly for assembly with MEDS oligo)
*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'''


===VI. Concentrate and adjust Tn5 molarity (optional)===
*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.
*1. Amicon Ultracel 30, 3000 rcf 20-25min periods to final OD280 >3.0 '''concentrated to OD ~7'''
*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'''
*2. If not used directly for transposase assembly, add 1.05 volume 99.5% glycerol and 0.33 volume of 2x Tn5 dialysis buffer (final 55% glycerol) and store at -20C




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added by us:
added by us:


*Tues: Transform C3013 bacterias and grow up plate overnight (amp selection)
*'''Tues:''' Transform C3013 bacterias and grow up plate overnight (amp selection)
*Wed: Inoculate 5 mL culture and grow up 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 5 mL culture to 1L culture, grow up, express protein, centrifuge, freeze -80 O/N
*'''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.
*'''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




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*Read OD280 vs dialysis buffer as blank.  
*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.
*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.




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'''6/10/2014'''
'''6/10/2014'''
*with 25 ng Jurkat DNA (unconcentrated transposase)
*with 25 ng Jurkat DNA (unconcentrated transposase)
*annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) for ~30 mins at RT
*annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30)
*tsp complex made by incubating at RT for ~30 mins
*reaction composition
*reaction composition
  2 uL 5X custom tagmentation buffer (D6)
  2 uL 5X custom tagmentation buffer (D6)
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'''6/11/2014'''
'''6/11/2014'''
*with 6 ng DNA (unconcentrated transposase)
*with 6 ng DNA (unconcentrated transposase)
*annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30) for ~30 mins at RT
*annealed sc1-T7tspn-NoP-bot to T7tspn-top2(10/30)
*tsp complex made by incubating at RT for ~30 mins
*reaction composition
*reaction composition
  1 uL 5X tagmetnation buffer
  1 uL 5X tagmetnation buffer

Latest revision as of 22:26, 17 July 2014

Tn5 production protocol and validations[edit]

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

File:ZhangLab 2 2014-06-11 SDS-PAGE gel tn5.png