Team:MIT/Repressor Group Experiment 1 Midi Preps

• 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: Midiprep

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Maya Kaul

Date: 2016-06-06

Monday, 6/6/16

Goal:

What We Did:

results:

	A	B
1	DNA	concentration (ng/uL)
2	hef1a/rtTa	724.5
3	tal 14/eyfp	1326.7
4	TRE/Tal14	1757.5

Table1

output and location:

2mL tubes with DNA, in -20 freezer

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.

Midiprep 7.1.16

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Elizabeth Strand

Date: 2016-07-11

Monday, 7/11

Midiprep

Introduction

Purifying large amount of DNA to tranfect mammalian cells

Materials

• Qiagen MidiPrep

Procedure

Midiprep for Tal21:EYFP, TRE :Tal21, hef1a:Gal4, TRE:BM3R1, BM3R1:EYFP

Made with Benchling

Project: miRNA and Repressors subgroup

Authors: Wangui Mbuguiro

Date: 2016-07-13

Wednesday, 7/13

	A	B	C
1	Tube	Concentration (ng/ul)	Concentration for Sequencing
2	Tal21:EYFP	403.4	80
3	TRE :Tal21	826.2	80
4	hef1a:Gal4	376.6
5	TRE:BM3R1	429.3
6	BM3R1:EYFP	405.6

Table1