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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

DNA ligation

Each reaction of 20 μL contained:

1 μL T4 ligase*
2 μL T4 ligase buffer*
The cut DNA fragments
Sterile Milli-Q water up to 20 μL

The DNA was mixed in a molar ratio of between 3:1 and 5:1 (insert:vector), and the volume of DNA was dependent on the concentration of the fragments and the number of ligations requiring the same fragments. The reactions were incubated at RT for 2-3 hours. To assess the background vector self-ligation rate, we always included controls with no insert added. Furthermore we included controls with no insert, and no ligase to estimate the amount of uncut vector. The ligation products were immediately used to transform cells. * Thermo Scientific

Fluorometer measurement

A Shimadzu RF-5301PC Spectrofluorophotometer was used.

For Spinach measurements the instrument was configured with an excitation wavelength of 482 nm and an emission wavelength of 505 nm.
The excitation slit was set to 5 and the emission slit was set to 3. The sizes of the slits determine how wide a range of wavelengths is let through the excitation and emission monochromators. Thus a large slit will increase the signal, but will decrease the wavelength precision of the instrument. The fluorometers spectrum function was used to find the largest slits that did not allow significant amounts of excitation light to be detected at the emission wavelength.
A cuvette with pure water was used to blank the instrument.

A 10 ml sample of culture was centrifuged at 4000g for 5 min. The pellet was washed twice and resuspended in 1 ml 0.9% NaCl. The OD600 of the sample was then measured and DFHBI-1T was added to a final concentration of 200 μM. After 1 hour on ice, the fluorescence of the sample was measured.

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.

Glycerol stock-preservation

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

PCR

As the polymerase is temperature sensitive it should only be removed from the freezer for short periods of time, therefore add the polymerase as the final part of your mastermix. When the polymerase have been added to keep the mastermix on ice. When the mastermix is complete remember to mix it thoroughly by pipetting, do not vortex the mastermix as this can ruin the polymerase. The mastermix is aliquoted to individual PCR tubes and the tubes are transferred to a thermo cycler. The annealing temperature should be 5 °C below the melting temperature of the primers, this step can be tricky as most Tm predictors tend to disagree. Depending on polymerase the elongation time should be 30 seconds per kb of the longest expected fragment.

Plasmid purification

Plasmids were purified from a 2 mL LB culture with the antibiotic that is used as the plasmid’s selection marker, grown at 350 rpm and 37 °C overnight.
Good aeration is important for obtaining high plasmid concentrations, so tube inclination should optimally be 45 °.
The purification was done with a Zymo Zyppy Plasmid Miniprep Kit, by following the manufacturer’s manual.

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.

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.

RNA degradation rate

The strain producing Spinach was grown in 2 mL LB overnight.
This culture was used to inoculate 200 mL LB in a shake flask which was incubated at 37 °C for 4 hours.
Rifampicin was then added to a final concentration of 100 μg/mL, at t = 0. Samples of 10 mL were taken every hour:

Each sample was centrifuged, washed and resuspended in 1 mL 0.9 % NaCl.
The OD600 of the sample was measured.
DFHBI-1T was added to a concentration of 200 μM and incubated on ice for 1 hour.
The fluorescence of the sample was then measured on a fluorometer

Background fluorescence level from cells was established by measuring a similarly treated sample from a strain that did not produce spinach.

Spinach standard series

Spinach produced by in vitro transcription was mixed with DFHBI-1T in different known concentrations. The fluorescence of each sample was measured on a fluorometer. A buffer was added to a final concentration of:

20 mM HEPES
100 mM KCl
5 mM NaCl
100 μM MgCl₂

The samples were kept on ice to minimize temperature-dependent fluorescence variations.

Transformation of 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

Revision as of 12:48, 17 October 2014 (view source) Mosbech (Talk \| contribs) ← Older edit		Revision as of 12:49, 17 October 2014 (view source) Mosbech (Talk \| contribs) Newer edit →
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	</br>		</br>
-	As the polymerase is temperature sensitive it should only be removed from the freezer for short periods of time, therefore add the polymerase as the final part of your mastermix. When the polymerase have been added to keep the mastermix on ice. When the mastermix is complete remember to mix it thoroughly by pipetting, do not vortex the mastermix as this can ruin the polymerase. The mastermix is aliquoted to individual PCR tubes and the tubes are transferred to a thermo cycler. The annealing temperature should be 5 °C below the melting temperature of the primers, this step can be tricky as most Tm predictors tend to disagree. Depending on polymerase the elongation time should be 30 seconds per kb of the longest expected fragment.~~</li>~~	+	As the polymerase is temperature sensitive it should only be removed from the freezer for short periods of time, therefore add the polymerase as the final part of your mastermix. When the polymerase have been added to keep the mastermix on ice. When the mastermix is complete remember to mix it thoroughly by pipetting, do not vortex the mastermix as this can ruin the polymerase. The mastermix is aliquoted to individual PCR tubes and the tubes are transferred to a thermo cycler. The annealing temperature should be 5 °C below the melting temperature of the primers, this step can be tricky as most Tm predictors tend to disagree. Depending on polymerase the elongation time should be 30 seconds per kb of the longest expected fragment.

Number of Reactions:
MQ water	μL
5X HF buffer	μL
2mM dNTP	μL
Polymerase	μL
Template	μL
10μM Primer x2	μL

Team:DTU-Denmark/Methods/Protocols