Team:MIT/miRNA Group Notebook protocols old data

• 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

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

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:

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

00:10:00

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

Lipofectamine 2000 Optimization for DNA

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Wangui Mbuguiro

Dates: 2016-08-04 to 2016-08-12

Thursday, 8/4

Done in tHESC --> Scroll down to 2.0, Julia

Transfection marker: hef1a:mKate (109 ng/ul)

Reagent: Lipofectamine 2000

Independent Variable: Amount of Lipofectamine 2000 (1.0uL, 1.5 uL, 2.0 uL, 2.5 uL)

Dependent Variable: Amount of fluorescene

Control: Amount of DNA (500ng per well), 24 Well plate

Well 1: 1.0 uL lipofectamine

Well 2: 1.5 uL

Well 3: 2.0 uL

Well 4: 2.5 uL

Well 5: 1.0 uL lipofectamine '

Well 6: 1.5 uL '

Well 7: 2.0 uL'

Well 8: 2,5 uL'

Well 9: untransfected

Dilute lipofectamine 2000 reagent in opti-mem until total volume 50uL

Dilute DNA in opti-mem until total volume 50 uL

Wait 5 mins.

Combine 25uL of DNA and lipofectamine dilutions into one tube and incubate for 20 minutes.

Add the 50uL of DNA+Lipofectamine complex to seeded wells.

Setup:

Eppendorf tubes

Lipofectamine tubes:

2 uL Lipofectamine, 48 uL opti-mem

3 uL Lipofectamine, 47 uL opti-mem

4 uL Lipofectamine, 46 uL opti-mem

5 uL Lipofectamine, 45 uL opti-mem

DNA (hef1a:mKate) tubes:

10 uL hef1a:mKate, 40 uL opti-mem X 4

Checked 3 hours later: No fluorescence

Cyto Flo Results: Minial to no transfection

Friday, 8/12

Lipofectamine 2000 DNA Transfection Optimzation 2.0

Transfection marker: hef1a:mKate (109 ng/ul)

Reagent: Lipofectamine 2000

DNA: ANY (except hef1a:mKate). Could dilute to 100 ng/ul for ease

Independent Variable: Amount of Lipofectamine 2000 (3.0uL, 4 uL, 5 uL, 6 uL)

Dependent Variable: Amount of fluorescene

Well 1: 3.0 uL lipofectamine

Well 2: 4.0 uL

Well 3: 5.0 uL

Well 4: 6.0 uL

Well 5: untransfected

Dilute lipofectamine 2000 reagent in opti-mem until total volume 50uL

Dilute DNA in opti-mem until total volume 50 uL

Wait 5 mins.

Combine 25uL of DNA and lipofectamine dilutions into one tube and incubate for 20 minutes.

Add the 50uL of DNA+Lipofectamine complex to seeded wells.

Setup:

Eppendorf tubes

Lipofectamine tubes:

6 uL Lipofectamine, 44 uL opti-mem

8 uL Lipofectamine, 42 uL opti-mem

10 uL Lipofectamine, 40 uL opti-mem

12 uL Lipofectamine, 38 uL opti-mem

DNA (hef1a:mKate) tubes:

10 uL hef1a:mKate, 40 uL opti-mem X 4

Making SOC

Introduction

SOC = SOB ("Super Optimal Broth") + Glucose (@40% w/v) Brian advised having SOB0 autoclaved first, and then adding glucose to SOB using a vacuum + filter with proper aseptic technique. If you end up making SOC, please write up detailed procedures, so other people can too :)

Materials

• SOB
  • In coldroom or on bench
• 40% Glucose
  • Above bench or ask Brian

Procedure

• Dilute 40% glucose to 2% using SOB (1:20)

1. Use a sterile bottle (same kind that 40% glucose in)

2. This can be done at the bench

Midiprep

Introduction

Purifying large amount of DNA to tranfect mammalian cells

Materials

• Qiagen MidiPrep

Procedure