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Interlab Study



We are very excited to announce we've transformed our DNA parts for the 2017 iGEM Interlab Study. Check out our Twitter page for weekly updates on our project!

We used the DNA parts from Plate 7 in the iGEM distribution kit. We successfully completed all stages of the InterLab Study and submitted it on time.

♦Process Flow♦

 Steps with an asterisk are not required in the iGEM interlab study, rather they are control steps to preserve the DNA and ensure that if anything goes wrong, we have a stock to retransform

 We choose two colonies per plate so we can run biological duplicates. This can provide information on variability of compounds within a single organism (in this case, DH5a e. Coli)

 The iGEM plate reader protocol has a series of dilutions, so we will have to pay careful attention to those details when the time comes

 Form 1 can be filled out at any time, Form 2 requires spreadsheet data from plate reader, Form 3 is in regard to the culturing process

♦Protocols♦

LB Agar Plates
Introduction

Used in conjunction with appropriate antibiotics to selectively plate
Materials

 LB Agar (NOT agarose), 35g/L

 appropriate antibiotic (ie, for 800mL LB Agar, add 800uL Amp, or 400Cm)

Procedure

Stir bar before autoclave

 If stir bar is forgotten, sterilize in EtOH to flame before dropping in autoclaved LB agar

 Mix appropriate amount of agar and DI water, dissolving completely to prevent burning agar

 Autoclave on liquid cycle (15 min is fine)

 Immediately after completing autoclave, cool on stir plate until not too hot to hold

 Add antibiotic

 Don't degrade antibiotic, but don't let cool too much and harden

Chemical Transformation
Introduction

Use RuCl2 from -80 freezer
Materials

 100uL cells (thawed on ice)

 250uL sterile SOC

Procedure

Process will take approximately 1h45min

 Add DNA to thawed cells on ice

 30 minute incubation on ice for transformation

 42oC heat shock for 45 seconds

 1 minute on ice

 Add 250mL sterile SOC to 2mL tube

 Place in 37oC incubator at 250 RPM for 1 hour (recovery)

5mL Overnight
Introduction

Use this protocol for making permanent glycerol stocks, temporary glycerol stocks, and mini-prepping plasmid.
Materials

 5mL LB broth

 Antibiotic: 2.5 μL Chloramphenicol- Cam 50 (usually)

Procedure

Start 12-14 hours before commencing mini-prep

 Place in 37oC incubator at 250 RPM overnight

 CRISPR is a low-copy plasmid so 10-11mL overnights should be used, depending on whether or not glycerol stock is necessary

 If making glycerol stock, use 6mL LB broth so 800μL can be used for stock

Inoculation Protocol
Introduction

Make 16 5mL overnight cultures from selected colonies (2 per plate)
Materials

 Plates grown from transformations

 Serological pipette

 50mL disposable serological pipette tip

 15mL tubes with purple cap (you

 Sterile LB broth

 10uL pipette with orange tips and 200uL pipette with yellow tips

 Chloramphenicol

 Autoclaved bottle that can hold 90mL of LB

 70% ethanol

Procedure

1. Label tops and sides of purple capped tubes #1A-8A and #1B-8B

 1A, the first colony you select on plate 1

 1B, the second colony you select on plate 2

2. Mark colonies on plates

 Find 2 colonies that are isolated on each plate

 Circle on back like our previous round

3. Prepare media

A. The smartest way to do this is to make a stock solution of well mixed media with antibiotic and aliquot the stock solution to the 16 tubes

B. Stock solution

 We need 5mL per culture and are making 16 cultures. Plan for 18 to be on the safe side so additional media doesn’t need to be made.

 18*5=90mL

 Our antibiotic is either 1000x (Cm25) or 2000x (Cm50), depending which stock tube you grabbed. This means if you are using a tube of Cm25, you need 90uL of antibiotic and if you are using a tube of Cm50, you will need 45uL of antibiotic (as it’s twice as concentrated)

C. Sterilize gloves, bench, and serological pipette with 70% ethanol. Work by a lit Bunsen burner to leverage the updraft of potential contaminant microbes

D. Attach sterile serological pipet tip and use to aliquot 90mL into a clean, autoclaved bottle.

 **be very careful with the 1L bottle of stock LB broth as it does not have antibiotic and can be easily contaminated

E. Use 200uL pipet to add the appropriate amount of antibiotic (see step 3.A.iii)

F. Swirl jar to ensure mixing

G. Return antibiotic to the freezer

4. Using Serological pipette, aliquot 5mL of well-mixed media with antibiotic to each 15mL tube

 Be sure to work near the flame

5. Inoculation

 Sterilize bench, gloves, 10uL pipette (sterilize really well) and work near the flame

 Touch pipette tip to colony and eject into appropriate tube with LB broth

 Work slowly and carefully to prevent cross-contamination

6. Place into tube racks of shaking incubator and record approximate time of inoculation

7. Clean up bench, ethanol working space, and neatly stack plates

8. Wash stock bottle used for LB and antibiotic and place on rack to dry

Restriction Digestion
Introduction

DNA concentration should be precise
Materials

 500ng DNA

 1μL enzyme

 5uL 10x cutsmart buffer

 1X remainder milliQ

Procedure

Protocol

 Leave for at least an hour in 37oC stationary incubator

 If using NEB BsaI-HF, add CIP (CALF intestinal phosphatase) and leave for another hour at 37oC

♦Results♦