Team:MIT/Repressor Group Experiment 1 LR Reactions

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
  Parts Toolbox Accessible Protocols Integrated Human Practices Public Outreach Interlab Study
• NOTEBOOK
  Lab Notebook Parts Mammalian Parts Collection Attributions Safety
• AWARDS
  HP Silver HP Gold Integrated HP Basic Part
• TEAM

Experiment 1 LR Reactions

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Maya Kaul

Date: 2016-04-01

Friday, 4/1/16

Goal: Create pEXP_hef1a:rtTA, pEXP_TRE:TALER14, pEXP_Tl14:meYFP via stock plasmids and LR reaction

What we did:

●

TE buffer was not used for the reactions because the pDEST was at half the concentration desired. To compentsate for the lower concentration, we used double the volume of pDEST and no TE buffer.

Results:

●

LR reactions were stored at -20 degrees Celsius after the reactions were completed.

LR Reaction (4/1/2016, Liz, Wangui, Maya, Kathryn)

Introduction

An LR reaction insertd one or more parts in pENTR vectors into pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTER plasmif: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 5 fmol/ul
• 200 ul PCR strip tubes, 1 tube per rxn ( 3 tubes)
• 5x LR Clonase II
  • Stored in ~5 ul liquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 ul liquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following tabe:

	A	B	C	D
1	Tube Label	TE	TT	HR
2	Promoter pENTR	pTal14	pTRE	hef1a
3	Gene pENTR	mEYFP	Tal14	rtTA
4	pDEST
5		Liz	Wangui	Maya

Table1

2. For each LR, label a 200 ul strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 ul of promoter pENTR

• -- 1 ul of the gene pENTR

• -- 2 ul of the pDEST

4. Retrieve an aliquot of LR Clonase from the -80

• Bring an razor blade with you, you'll need to cut s tube from the strip tubes

5. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom

6. Add 1 ul of the LR clonase to each LR reaction.

7. Cap the tubes.

8. Flick them several times to mix.

9. Pulse-spin the tubes in the microfuge to collet the liquid at the bottom.

10. Incubate at room temperature for at least 12 hours and not more than 24 hours. (Started incubation at 4:30pm)

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and date

• 16-24 Hours later: Proteinase K kill (Saturday 10am)

11. Retrieve a 5 aliquot of peoteinase K from the -80 freezer

12. That in your fingers, then pulse in the microfuge to collect at the bottom of the tube

13. Pipette 1 ul into each of the LR reations

14. Flick serveral times to mix

15. Pulse-spin the tubes in the microcentrifuge

16. Incubate at 37 degrees Celsius for 15 minutes, or room-temperature for an hour:

00:15:00

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation

17. Proceed to transformation. Transform 2 ul. (Sunday 3pm)

Experiment 1 LR/transformation/miniprep for BM3R1 and Tal21-involved parts

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Maya Kaul

Date: 2016-06-14

Tuesday, 6/14/16

Transformation of E. coli

Introduction

Transformation is the process of inducing chemically competent E. coli to take up DNA.

Materials

• Dry bath, set to 42°C
  • Fill the wells in the dry bath block 1/2 full with DI water.
• Ice bucket, with ice
  • For thawing competent cells.
• DNA to transform
  • Could be an assembly reaction (LR, Golden Gate, etc) or a miniprepped plasmid.
  • If you removed it from the freezer, make sure it's entirely thawed out.
• pUC19 Transformation Control, 1 pg/µl
  • The pUC19 control will tell you how efficient your transformations were.
• SOC growth media, at room temperature
  • Check to make sure it's clear and NOT CLOUDY.
• Antibiotic plates, one per transformation, plus 1 Amp plate for the pUC19 control
  • Make sure the plates you use match the resistance cassette of the plasmid!
• Competent E. coli, one tube per transformation + one for the pUC19 control
  • These live in the -80 in 235.
  • Thaw on ice 3-4 minutes.
• A timer, set for 30 seconds.

Procedure

• Setup

1. Make sure the dry bath is set to 42°C and the wells in the block are 1/2 full of DI water

2. Remove selection plates from the refrigerator. Double-check that they match the selection marker on your plasmid, then place them in the 37° incubator.

3. Retrieve the DNA to transform.

• If frozen: thaw, completely, flick a few times to mix, then pulse down in the microfuge.

4. Fill an ice bucket with ice. Retrieve one tube of competent E. coli per transformation from the -80 and thaw on ice, 3-4 minutes.

5. While the transformation tubes are thawing, label their tops with something descriptive. Record the labels here:

	A	B	C	D	E	F	G	H
1	TRE: Tal21 TT21	hef1a: Gal4 HG	Tal: eYFP TE	TRE: BM3R1 TB	BM3R1: eYFP BE	puc19
2

Table1

• Transformation

6. Add 2 µl DNA from each reaction to a tube of competent cells.

• Immediately after adding the DNA to each tube, stir the cells a few times with the pipette tip.

7. Add 1 µl of the pUC19 transformation control to the positive control tube.

8. Incubate on ice for 30 minutes.

00:30:00

9. Heat shock the cells for exactly 30 seconds in the 42° heat block. (Yes, set a timer.)

00:00:30

10. Place back on ice for 2 minutes.

00:02:00

11. Add 250 µl SOC to each tube.

12. Tape the tubes to the platform of a shaker at 37°C and shake at 270 RPM for 60 minutes.

• Plating

13. Label the selection plates using the labels you recorded above.

14. Shake ~10 plating beads onto each plate.

15. Pipette 100 µl of each transformation onto the corresponding plates.

16. Cover the plates and shake the beads around to spread the cells out.

17. Dispose of the beads by tapping them into the waste container.

18. Incubate the plates upside down overnight in the 37° incubator.

• Don't incubate for more than 18-24 hours.

• Compute transformation efficiency

19. Count the colonies on your positive transformation plate.

• If there are many many colonies, then hooray! You had a great transformation. Just estimate.

20. Divide the number of colonies by the fraction of the transformation you plated.

• So, if you resuspended your transformation in a total volume of 300 ul, then plated 100 ul, multiply the number of colonies by 3.

21. Transformation efficiency is expressed in colonies per microgram pUC19. Multiply the number of colonies by the appropriate conversion factor.

• So if you transformed 1 picogram of pUC19 DNA, multiply by 10⁶.

22. Record your transformation efficiency in your (daily) lab notebook.

LR Reaction

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTR plasmid: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 10 fmol/ul
• Nuclease-free TE
• 200 µl PCR strip tubes, 1 tube per rxn
• 5x LR Clonase II
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label	12
2	Promoter pENTR	L4 TRE tight R1	L4 TRE tight R1
3	Gene pENTR	L1 BM3R1 L2	L1 Taler21 L2
4	pDEST	L4-pDEST-L2	L4-pDEST-L2
5
6

Table1

2. For each LR, label a 200 µl strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 2 µl of the pDEST

4. Retrieve an aliquot of LR Clonase from the -80.

• Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

5. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

6. Add 1 µl of the LR clonase to each LR reaction.

• Be careful pipetting; LR clonase is viscous.

7. Cap the tubes.

8. Flick them several times to mix.

9. Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

10. Incubate at room temperature for at least 12 hours and not more than 24 hours.

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

• 16-24 hours later: Proteinase K kill

11. Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.

12. Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

13. Pipette 1 ul into each of the LR reactions.

14. Flick several times to mix.

15. Pulse-spin the tubes in the microcentrifuge.

16. Incubate at 37° for 15 minutes, or room-temperature for an hour.

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

17. Proceed to transformation. Transform 2 µl.

18. Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

LR Reaction: Tal21,

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTR plasmid: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 10 fmol/ul
• Nuclease-free TE
• 200 µl PCR strip tubes, 1 tube per rxn
• 5x LR Clonase II
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label	hg	Tt	Be	Te
2	Promoter pENTR	L4_hef1a_R1	L4_TRE_R1	L4_BM3R1_R1	L4_Tal21_R1
3	Gene pENTR	L1_gal4VP16_L2	L1_Tal21_L2	L1_eYFP_L2	L1_eYFP_L2
4	pDEST
5
6

Table1

2. For each LR, label a 200 µl strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 1 µl of the pDEST

4. Add 1 µl of TE to each tube

5. Retrieve an aliquot of LR Clonase from the -80.

• Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

6. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

7. Add 1 µl of the LR clonase to each LR reaction.

• Be careful pipetting; LR clonase is viscous.

8. Cap the tubes.

9. Flick them several times to mix.

10. Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

11. Incubate at room temperature for at least 12 hours and not more than 24 hours.

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

• 16-24 hours later: Proteinase K kill

12. Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.m n

13. Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

14. Pipette 1 ul into each of the LR reactions.

15. Flick several times to mix.

16. Pulse-spin the tubes in the microcentrifuge.

17. Incubate at 37° for 15 minutes, or room-temperature for an hour.

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

00:15:00

18. Proceed to transformation. Transform 2 µl.

19. Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

Overnight liquid cultures (picking colonies)

Introduction

Overnight cultures are used to prepare miniprep DNA.

Materials

• The plate from which you are picking colonies
• 15 ml round-bottom polystyrene tubes, one per culture
  • The ones with the snap caps, NOT conical tubes with screw caps
• 5 mL LB per culture
• A container that can hold 5ml x the number of cultures
  • For a modest number of minipreps, a 50 ml conical tube works well.
  • For larger minipreps, use a sterile bottle (100 ml is frequently useful.)
• Antibiotic stock, 1000X

Procedure

• Materials Setup

1. Warm up the LB to at least room temperature (if it came from the fridge), but not warmer than 37°C

2. Label one round-bottom culture tube for each miniprep. Use "NAME-1, NAME-2, ..."etc for the naming convention, where NAME is a shortened name of the plasmid (eg, "hEF1a:mKate").

• Your impulse is to just use number, or initials and number, but trust me -- you will want to be able to identify this tube in three weeks when you've forgotten what you were doing.

3. Using a sterile pipette, transfer 5 ml of LB to the mixing container for each culture PLUS 5 ML.

4. Add antibiotic stock to a final concentration of 1X (1 µl stock for each 1 ml in the mixing container.)

5. Cap tightly and mix well.

• Culture Setup

6. Using a sterile pipette, transfer 5 ml of LB+antibiotic to each round-bottom culture tube.

• If you are making cultures with different antibiotics, take care that the right media goes in each tube.

7. Squirt ethanol on a pair of foreceps and wipe dry with a Kimwipe.

8. Use the foreceps to pick up a sterile 200µl pipette tip, scrape a colony off of the plate, and drop the pipette tip in the corresponding tube.

• Repeat for each tube.

9. Transfer to an incubating shaker at 37°C and incubate 14-16 hours.

• Don't over-grow too badly, or your yield will suffer.

• If you need to grow longer, you can grow at 30°C instead for 20 hours.

Miniprep

Introduction

The miniprep uses silica gel to isolate plasmid DNA from an E. coli culture

Materials

• Buffer P1 (resuspension buffer)
  • Retrieve from refrigerator. If you are opening a new miniprep kit, add the RNAse and LyseBlue reagent and check the box on the cap.
• Buffer P2 (lysis buffer)
  • Open the cap and look at the lysis buffer. Swirl it around. If it appears cloudy, the SDS has fallen out of solution; warm it for a few minutes in the 55°C water bath.
• Buffer N3 (neutralization buffer)
• Buffer PB (binding buffer)
• Buffer PE (rinse buffer)
  • Make sure the "Ethanol added?" box has been checked. If you are opening a new miniprep kit, add absolute ethanol as per the kit instructions and check the box on the cap.
• Buffer EB (elution buffer)
• Miniprep waste container
  • Miniprep buffers contain salts that can't go down the sink.
• Per miniprep: two microcentrifuge tubes and one blue spin column, with collection tube.

Procedure

• Harvest and resuspension

1. For each culture, label two microcentrifuge tubes on the cap and one blue spin column on the side.

• The spin columns should be in their (cap-less) collection vials.

2. Pipette 1.8 mL of each culture into the corresponding microcentrifuge tubes

3. Centrifuge at maximum speed (10,000 or 13,000xg) for three minutes.

4. Aspirate the supernatant, or pour it off into the bleach bucket.

5. Pipette ANOTHER 1.8 ml of each culture into the corresponding microcentrifuge tubes.

6. Centrifuge at maximum speed for three minutes.

7. While the centrifuge is running, move the remaining cultures to 4degC.

8. Aspirate the supernatant off with the bench aspirator. Be careful not to disturb the pelleted E. coli.

• We use an aspirator here because the less extra salt and protein we put in the miniprep, the better the yield is.

9. Add 250 µl Buffer P1 to each tube.

10. Resuspend the E. coli pellet. The preferred way is with the roto-mixer at the other end of the lab.

• Alternately, if you have just a few tubes, you can resuspend on a vortex.

• Make sure to resuspend fully and thoroughly. The resulting suspension should be smooth and cloudy; if there is particulate matter floating around, vortex some more.

• Lysis

11. Add 250 µl Buffer P2 to each tube.

• Work quickly; the lysis step should take less than 5 minutes.

12. Snap the tubes closed and invert them 4-6 times, until the tube is thoroughly mixed and the entire solution turns blue.

• If you have many many tubes, you can stack a second tube rack on top of them and invert the entire thing.

13. Add 350 ul Buffer N3 to each tube.

14. Snap the tubes closed and invert 4-6 times, until the solution is thouroughly mixed and no longer blue.

• The solution will become cloudy or flocculent.

15. Centrifuge on high speed for 10 minutes.

• Separation

16. Remove the tubes from the microcentrifuge, being careful not to disturb the white pellet.

17. Using P-1000 micropipettor set to 850 ul, carefully transfer the supernatant from each centrifuge tube to the corresponding blue spin column.

18. Centrifuge the spin columns for 30 seconds at maximum speed.

• Don't forget to put the lid on the rotor! Some of the salts get aerosolized because the spin columns don't have caps.

19. Pour the flow-through from each column into the miniprep waste container.

20. Pipette 500 ul of Buffer PB onto each spin column.

21. Centrifuge the spin columns for 30 seconds at maximum speed.

22. Pour the flow-through from each column into the miniprep waste container.

23. Pipette 750 ul of Buffer PE onto each spin column

24. Wait 1-3 minutes.

• This allows some of the salt that's still bound to the silica matrix to resuspend in the buffer.

25. Centrifuge the spin columns for 30 seconds at maximum speed.

26. Pour the flow-through from each column into the miniprep waste container.

27. Return each spin column to its collection tube and centrifuge an additional 1 minute at high speed.

• This removes every last trace of buffer PE; the ethanol can screw up downstream steps.

28. Transfer each spin column to a clean labelled microcentrifuge tube.

29. Pipette 50 ul of Buffer EB onto the center of each column.

• The volume of EB is comparable to the volume of silica gel matrix; if you pipette down the side, you might not get the entire transfer to the matrix.

30. Wait 1-3 minutes.

• This gives the DNA a chance to dissociate from the silica matrix.

31. Centrifuge the spin columns, in their collection tubes, for one minute at maximum speed.

32. Proceed directly to analyze the samples on the Nanodrop.

Nanodrop

Introduction

Use the nanodrop to measure the DNA concentration in a sample.

Materials

• The DNA to measure
• Buffer EB (Elution Buffer)
  • Usually found at the Nanodrop station.

Procedure

• Blank the Nanodrop

1. If it's not running, start the Nanodrop 2000 software. Select "Nucleic Acids."

2. Ensure that the Type drop-down box on the right-hand side reads DNA.

3. Ensure that the Use cuvette box on the left-hand side is off.

4. Raise the Nanodrop arm.

5. Squirt a Kimwipe with a little water and gently wipe off both the measurement surfaces (the pedestal and the light aperture.)

6. Use a dry Kimwipe to gently wipe off both measurement surfaces.

7. Pipette 1.5 ul of Buffer EB onto the pedestal.

8. Gently lower the Nanodrop arm.

9. Click the Blank button. Wait a few seconds for the instrument to blank.

• Measure your samples

10. Gently wipe off both measurement surfaces.

11. Pipette 1.5 ul of your sample onto the pedestal.

12. Lower the Nanodrop arm.

13. Click the Measure button.

14. Record the concentration on the side of the tube and in the plasmid's notebook page.

15. Gently wipe off both measurement surfaces.

• You do not need to use water to clean the surfaces between measurements; the measurement surfaces are hydrophobic and there is very littie sample careover.

16. Repeat steps 11-15 for each sample.

• Clean the Nanodrop

17. Squirt a little water on a dry Kimwipe and wipe off both measurement surfaces.

18. Use a dry Kimwipe to wipe off both measurement surfaces.

19. Lower the arm of the Nanodrop before walking away from the instrument.

LR Reaction for hef1a:Gal4VP16, BM3R1:meYFP and Tal21:meYFP

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Gizem Gumuskaya

Date: 2016-06-27

Monday, 6/27

Goal: Create hef1a:Gal6VP16, BM3r1:meYFP, Tal21:meYFP

LR Reaction

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTR plasmid: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 10 fmol/ul
• Nuclease-free TE
• 200 µl PCR strip tubes, 1 tube per rxn
• 5x LR Clonase II
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label	Tal21:meYFP	BM3R1:meYFP	hef1a:Gal4VP16
2	Promoter pENTR	L4_Taler21_R1	L4_BM3R1_R1	L4_hef1a_R1
3	Gene pENTR	L1_meYFP_L2	L1_meYFP_L2	L1_Gal4VP16_L2
4	pDEST	pDEST-T_R4_GTW6_R2	pDEST-T_R4_GTW6_R2	pDEST-T_R4_GTW6_R2
5
6

Table1

2. For each LR, label a 200 µl strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 1 µl of the pDEST

4. Add 1 µl of TE to each tube

5. Retrieve an aliquot of LR Clonase from the -80.

• Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

6. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

7. Add 1 µl of the LR clonase to each LR reaction.

• Be careful pipetting; LR clonase is viscous.

8. Cap the tubes.

9. Flick them several times to mix.

10. Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

11. Incubate at room temperature for at least 12 hours and not more than 24 hours.

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

• 16-24 hours later: Proteinase K kill

12. Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.m n

13. Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

14. Pipette 1 ul into each of the LR reactions.

15. Flick several times to mix.

16. Pulse-spin the tubes in the microcentrifuge.

17. Incubate at 37° for 15 minutes, or room-temperature for an hour.

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

00:15:00

18. Proceed to transformation. Transform 2 µl.

19. Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

LR Reaction for TRE:BM3R1 and TRE:Tal21

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Gizem Gumuskaya

Date: 2016-06-29

Wednesday, 6/29

LR Reaction

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTR plasmid: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 10 fmol/ul
• Nuclease-free TE
• 200 µl PCR strip tubes, 1 tube per rxn
• 5x LR Clonase II
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label	TRE:BM3R1	TRE:Taler21
2	Promoter pENTR	TRE_Tight	TRE_Tight
3	Gene pENTR	BM3R1	Taler21
4	pDEST	pDEST
5		T:B	T:T21
6

Table1

2. For each LR, label a 200 µl strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 1 µl of the pDEST

4. Add 1 µl of TE to each tube

5. Retrieve an aliquot of LR Clonase from the -80.

• Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

6. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

7. Add 1 µl of the LR clonase to each LR reaction.

• Be careful pipetting; LR clonase is viscous.

8. Cap the tubes.

9. Flick them several times to mix.

10. Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

11. Incubate at room temperature for at least 12 hours and not more than 24 hours.

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

• 16-24 hours later: Proteinase K kill

12. Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.m n

13. Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

14. Pipette 1 ul into each of the LR reactions.

15. Flick several times to mix.

16. Pulse-spin the tubes in the microcentrifuge.

17. Incubate at 37° for 15 minutes, or room-temperature for an hour.

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

00:15:00

18. Proceed to transformation. Transform 2 µl.

19. Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

Tal14:EYFP

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-07-14

Thursday, 7/14

Replacing Tal14:meYFP with Tal14:EYFP in our stocks since Brian suggested that we use EYFP instead of meYFP.

LR Reaction

Introduction

An LR reaction inserts one or more parts in pENTR vectors into a pDEST vector. Used to assemble transcriptional units from promoters and genes.

Materials

• Promoter pENTR plasmid: L4-Promoter-R1
  • Working concentration: 5 fmol/ul
• Gene pENTR plasmid: L1-Gene-L2
  • Working concentration: 5 fmol/ul
• Destination plasmid: pDEST
  • Working concentration: 10 fmol/ul
• Nuclease-free TE
• 200 µl PCR strip tubes, 1 tube per rxn
• 5x LR Clonase II
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.
• Proteinase K
  • Stored in ~5 µl aliquots in the -80 in room 235. Don't remove an aliquot until you're ready to use it.

Procedure

• LR Reaction Setup

1. For each LR you are doing, fill out a column in the following table:

	A	B	C	D	E	F
1	Tube Label
2	Promoter pENTR
3	Gene pENTR
4	pDEST
5
6

Table1

2. For each LR, label a 200 µl strip tube with your initials and tube number.

3. Into each tube, pipette:

• -- 1 µl of the promoter pENTR

• -- 1 µl of the gene pENTR

• -- 1 µl of the pDEST

4. Add 1 µl of TE to each tube

5. Retrieve an aliquot of LR Clonase from the -80.

• Bring an razor blade with you, you'll need to cut a tube from the strip tubes.

6. Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

7. Add 1 µl of the LR clonase to each LR reaction.

• Be careful pipetting; LR clonase is viscous.

8. Cap the tubes.

9. Flick them several times to mix.

10. Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

11. Incubate at room temperature for at least 12 hours and not more than 24 hours.

• A popular strategy is to tape the tubes to the shelves over the bench, with your initials and the date.

• 16-24 hours later: Proteinase K kill

12. Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.m n

13. Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

14. Pipette 1 ul into each of the LR reactions.

15. Flick several times to mix.

16. Pulse-spin the tubes in the microcentrifuge.

17. Incubate at 37° for 15 minutes, or room-temperature for an hour.

• PAUSE POINT: You can store the reactions in the -20 indefinitely until the transformation.

00:15:00

18. Proceed to transformation. Transform 2 µl.

19. Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)