Difference between revisions of "Team:UCLA/Notebook/Protein Cages/2 July 2015"

Latest revision as of 00:12, 3 July 2015

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Phillip's notes:

Introduction: Today, the plate will be checked for the presence of colonies. If they are present, then a liquid culture will be started in order to grow more of the Yeate’s bacterial strain. Aliquots will be taken and glycerol stocks will be made for use in the future. In addition, colony PCR may be done in order to verify that the plasmid of interest is still in the bacteria that grew.

There were colonies on the plates!

Observation: Much of the colonies were lawns, as the entire tube was used to plate yesterday. There were several individual colonies that grew from the later streaks.

Notes for growing E. coli from Qiagen website:

Growth of E. coli strains

Good microbiological technique will always ensure the best yield and quality of plasmid DNA. To prepare the perfect bacterial culture for your plasmid prep, follow the steps below.

1. Prepare a starter culture by inoculating a single colony from a freshly streaked selective plate into 2–10 ml LB (Luria-Bertani) medium containing the appropriate antibiotic. Grow at 37°C for ~8 hours (logarithmic growth phase) with vigorous shaking (~300 rpm).

Tip: Do not inoculate directly from glycerol stocks, agar stabs, or plates that have been stored for a long time, as this may lead to loss or mutation of the plasmid.

Tip: It is often convenient to grow the starter culture during the day so that the larger culture can be grown overnight for harvesting the following morning.

2. Dilute the starter culture 1/500 to 1/1000 into a larger volume of selective LB medium, as indicated in the appropriate plasmid purification protocol.

Use a flask of at least 4 times the volume of culture to ensure sufficient aeration. Do not use a larger culture volume than recommended in the protocol, as this will result in inefficient lysis and reduce the quality of the preparation.

3. Grow the culture at 37°C with vigorous shaking (~300 rpm) for 12–16 hours (see next section).

4. Harvest the bacterial culture 12–16 hours after inoculation. This corresponds to the transition from logarithmic into stationary growth phase, when cell density is high (3–4 x 109 cells per ml) and RNA content of cells is low. Harvesting too early may result in lower than expected yields of plasmid DNA due to a lower cell density. Harvesting too late may result in low plasmid quality and yield due to DNA degradation from over-aging of the culture.

Tip: Growth of cultures is dependent on factors such as host strain, plasmid insert and copy number, and culture medium. To determine the optimal harvesting time for a particular system, monitor the cell density and the growth of the culture by measuring the OD600 (see next section).

5. Harvest the bacterial culture by centrifugation at 6000 x g for 15 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube until all of the medium has been drained. The cells are now ready for the lysis procedure, as indicated in the appropriate plasmid purification protocol. The procedure may be stopped at this point and continued later by freezing the cell pellets obtained by centrifugation. The frozen cell pellets may be stored at –20°C for several weeks.

For storing E.coli strains:

Glycerol stocks

E. coli strains can be stored for many years at –70°C in 15% glycerol.

Prepare glycerol stocks of bacteria as follows:

1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving. Tip: Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.

2. Add 0.85 ml of a logarithmic-phase E. coli culture to the vial of pre-sterilized glycerol.

3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol.

4. Freeze in ethanol–dry ice or liquid nitrogen and store at –70°C.

Tip: Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria.

Tip: For precious strains, storage of 2 stock vials is recommended.

Tip: When recovering a stored strain, it is advisable to check the antibiotic markers by streaking the strain onto a selective plate.

Agar plates

Plates of streaked bacteria can be sealed with Parafilm and stored upside-down at 4°C for several weeks. Bacteria should always be streaked onto plates containing the appropriate antibiotic to ensure that selective markers are not lost.

To obtain well-isolated colonies, streak an agar plate as follows:

1. Flame a wire loop, and cool on a spare sterile agar plate.

2. Using the wire loop, streak an inoculum of bacteria (from a glycerol stock, stab culture, or single colony on another plate) across one corner of a fresh agar plate, as shown in the figure Streaking bacteria on agar plates.

3. Flame and cool the wire loop again. Pass it through the first streak and then streak again across a fresh corner of the plate.

4. Repeat again to form a pattern.

5. Incubate the plate upside down at 37°C for 12–24 hours until colonies develop.

Colony PCR notes from http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf :

Typical colony PCR reaction Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice).

38 µl sterile distilled water

5 µl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X 100)

3 µl 25 mM MgCl2

1 µl 10 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP)

1 µl 20 µM forward primer

1 µl 20 µl reverse primer

0.2-1 µl Taq polymerase

50 µl total volume

To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields.

PCR conditions

1 cycle : 5 min at 95°C -initial cell breakage and DNA denaturation

30-40 cycles: 1 min at 95°C -DNA denatures into single strands 30-40 cycles 1.5 min at 54°C -primers anneal to ssDNA template (temp depends on primers) 1 min at 72°C -primes are extended from 3'-end by Taq(1 min/kb)

1 cycle : 5 min at 72°C -final extension to make sure all products are full length (72°C is optimal for Taq polymerase)

Lab notebook:

Inoculation of E. coli

A starter culture tube was obtained, and 5mL of LB + 5uL of ampicillin was added.

To inoculate: Unscrew the LB bottle and flame the lid. Flame the lid of the culture tube and add the LB using a serological pipette. Cover everything when not in use, work quickly and near the flame.

Vinson helped with the streaking of the bacterial colony. The pipette was sprayed with ethanol, and the tip used was sterile. A colony was gently swept with the tip, and ejected into the culture tube. The tube began shaking at 3:30PM at 37 degrees.

Conclusions: Colony PCR was not done as primers were not ready for them. Tyler will take care of making the glycerol stocks tomorrow morning.

Today Phil looked over a lot of protocols for stuff we need to do over the next week or so. We innoculated a starter broth after picking a colony from our plate. Tomorrow I will create glycerol stocks to use early next week.

Tyler Lee --Wtleeiv 18:28, 2 July 2015 (CDT)