From 2014.igem.org

Revision as of 13:03, 15 October 2014 by Marktoe (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Protocols & Recipes
During our laboratory work several protocols and recipes were applied. To make sure that the work is documented in a systematical and reproducible manner, we have made a list of the procedures and added it to this page.

Protocols

Glycerol stock-preservation

Add one mL sterile 50% glycerol to a cryotube
Add one mL overnight culture to the cryotube
Store at -80 °C

PCR

Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
Mark the PCR tubes on lid and side and place in thermocycler.
The annealing temperature should match the primer melting temperatures minus 5 degrees. This can be hard since Tm predictors tend to disagree.
Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

Gel electrophoresis

1% agarose in 1x TAE buffer is melted and poured into a mold. Ethidium bromide is added to a concentration of 25 μL/L, and is mixed using a pipette tip. (Note: Ethidium bromide is a carcinogen, therefore use gloves when working with the gel. Remove the glove immediately after your work with ethidium bromide in order to avoid contamination of the lab).
A comb is placed in the mold to create wells when the agarose solidifies.

When the gel is set, the comb is removed and the gel is transferred to a vessel containing 1x TAE buffer. A suitable DNA ladder is added to the first well, and each sample is then added to an individual well.

When all samples have been loaded, the gel is run with a constant voltage usually 75-100 V for approximately 30 minutes. A lower voltage and a longer duration generally yields better separation.

The gel is analysed with UV light. The gel can then be photographed or bands can be cut out for further work.

Preparation of Cam stock solution

Recipe on M9 minimal media

Make M9 salts
To make M9 Salts aliquot 800ml H2O and add

64g Na2HPO4-7H2O
15g KH2PO4
2.5g NaCl
5.0g NH4Cl
Stir until dissolved
Adjust to 1000ml with distilled H2O
Sterilize by autoclaving

Measure ~700ml of distilled H2O (sterile)
Add 200ml of M9 salts
Add 2ml of 1M MgSO4 (sterile)
Add 20 ml of 20% glucose (or other carbon source)
Add 100ul of 1M CaCl2 (sterile)
Adjust to 1000ml with distilled H2O

Restriction digestion

Each reaction should contain:
- 1X NEBuffer (according to the desired restriction enzyme(s)) with BSA.
- 1 unit/μl restriction enzyme.
- The DNA that is to be cut, final concentration of 10 ng/μL
- Milli-Q water to a volume of 20 μL
Incubate the reactions at the restriction enzymes’ optimal temperature. If the restriction reaction is analytical, 30 minutes is enough. If the DNA is to be purified, a longer duration is recommended, e.g. 3 hours.
Add loading buffer to samples and run on 1% agarose gel with ethidium bromide (0.2 μg/ml).
Analyze your gel, and cut bands out if desired.

Preparation of competent cells

2 mL LB is inoculated with cells from a single colony and incubated at 37 °C O/N at 350 rpm
200 mL of preheated (37 °C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 °C and 350 rpm until OD 600 reaches 0.3 - 0.6
When OD600 reaches 0.3 - 0.6 the shake flask is chilled in an ice-bath until the culture is completely cooled. All steps from here should be kept as cold as possible.
The 200 mL culture is transferred to 50 mL tubes and spun at 5500 G for 10 minutes. The supernatants are discarded and each pellet is resuspended in 25 mL ice cold 0.1 Molar CaCl₂. The tubes are poured together to 2x50 mL.
The cells are spun at 5500 G for 10 minutes and the supernatant is discarded. The cells are resuspended in 1 mL 0.1 Molar CaCl₂ and transferred to microcentrifuge tubes.
The cells are spun at 15000 G for 2 minutes and the supernatant is discarded. The cells are resuspended in 1 ml 0.1 Molar CaCl2 with 15% glycerol.
The cells are aliquoted into portions of 200 μL in pre-chilled Eppendorf tubes and immediately stored at -80 °C.

Transformation with chemically competent E. coli

Transformation with chemically competent E. coli

IMPORTANT: Keep everything on ice unless otherwise stated in the protocol.

Competent cells are defrosted in an ice bath
50 μL cells is transferred to a cooled eppendorf tube
1-5 μl DNA is transferred to the competent cells, mixed by gently swirling the tube
DNA and cell mix is kept on ice for 10 minutes
Cells are heat shocked at 42 °C for 30 seconds in water bath
Cells are kept on ice for 3 minutes.
500 μL LB without antibiotics is added and incubated at 37 °C for 30-120 minutes depending on antibiotic marker
Plate cells on LB plate with the relevant antibiotic and incubate overnight at 37 °C

In vitro transcription

To prevent RNase, work in an RNase free environment:

Use gloves
Use pipettes dedicated to RNase-free work
Use RNase-free microcentrifuge tubes
Use RNase Zap to clean the bench

To obtain the DNA template for the transcription we used PCR amplification

The T7 promoter was added as a tail to the forward primer
The T7 promoter used was BBa_R0085
The PCR product was purified with a Qiagen kit and eluted in DEPC-treated water

Each transcription reaction mix consisted of:

20 μL Transcription Buffer
5 μL ATP 10mM
5 μL CTP 10mM
5 μL GTP 10mM
5 μL UTP 10mM
150 U T7 RNA polymerase
1 μg template DNA
DEPC-treated water to 100 μL

The reactions were incubated at 37°C for 2-3 hours, and stopped with the addition of 3 μL EDTA. The product was purified with Qiagen RNeasy Kit, and eluted in 50 μL DEPC-treated water.

Microscopy

Microscopy was used to visualize fluorescent cells, both with GFP and Spinach.

Cells are grown O/N in LB media to OD600 of approximately 4
Cells are washed in PBS and resuspended in M9 media with 200 μM DFHBI-1T
1 μl cell suspension is placed on a poly-lysin coated microscope slide and covered with a coverslide
Cells were visualized with 630x magnification using a GFP filter and UV light

Recipes

test3