Protocols and Other Methods Used

BioLine Isolate Genomic DNA Mini Kit:

1. Pellet cell culture, decant, add 1mL CLS-TC
2. Resuspend and transfer to Fastprep tube (BIO101)
3. Fastprep 5.5g for 30 seconds, then spin @ 14,000 rpm for 5 minutes
4. Recover 700uL then 700uL Binding Matrix
5. Rotate for 5 minutes then spin for 1 minute
6. Decant the supernatant and add 800uL SEWS then vortex
7. Rotate for 5 minutes then spin for 1 minute
8. Decant the supernatant then pulse spin and remove the last bit of ethanol
9. Air dry for 5 minutes then resuspend in 200uL TE buffer
10. Spin at 14,000 rpm for 3 minutes and recover 160uL

Promega Wizard® Plus SV Minipreps DNA Purification System:

1. Centrifuge bacterial culture at 12,000 rpm for 5 minutes, pour off the supernatant and blot the inverted tube on a paper towel to remove excess media
2. Add 250uL of Cell Resuspension Solution and completely resuspend the pellet by pippetting
3. Add 250uL of Cell Lysis Solution and mix by inverting tube 4 times, incubate for no more than 5 minutes
4. Add 10uL Alkaline Protease Solution and mix by inverting tube 4 times, incubate at room temperature for no more than 5 minutes
5. Add 350uL of Neutralization Solution and immediately mix by inverting the tube 4 times
6. Centrifuge the bacterial lysate at 14,000 rpm in a microcentrifuge for 10 minutes at room temperature
7. Transfer cleared lysate to Spin Column by decanting without disturbing precipitate
8. Centrifuge the supernatant at 14,000 rpm for 1 minute at room temperature, discard flowthrough
9. Add 750uL of Column Wash Solution (diluted with 95% ethanol), to Spin Column
10. Centrifuge at 14,000 rpm for 1 minute at room temperature, discard flow through
11. Add 250uL of Column Wash Solution
12. Centrifuge at 14,000 rpm for 2 minutes at room temperature
13. Transfer the Spin Column to a new tube
14. Elute the plasmid DNA by adding 100uL of Nuclease Free Water to the Spin Column
15. Centrifuge at 14,000 rpm for 1 minute at room temperature
16. Add 11uL 10x TE buffer for DNA storage at 4C

Promega pGEM®-T Easy Vector System:

(Ligation Using 2x Rapid Ligation Buffer)

1. Briefly centrifuge the pGEM®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube. Calculate the amount of insert DNA required to give a 1:3 ratio of vector to insert
2. Set up ligation reactions as described below. Vortex the 2x Rapid Ligation Buffer vigorously before each use. Use 0.5mL tubes known to have low DNA-binding capacity.

3. Mix the reactions by pipetting. Incubate the reactions 1 hour at room temperature. Alternatively, incubate the reactions overnight at 4C for the maximum number of transformants

1. Prepare LB/Ampicillin/IPTG/X-Gal plates
2. Centrifuge the ligation reactions briefly. Add 2uL of each ligation reaction to a sterile 1.5mL tube on ice. Prepare a control tube with 0.1ng of uncut plasmid
3. Place the JM109 High-Efficiency Competent Cells in an ice bath until just thawed (5 minutes). Mix cells by gently flicking the tube
4. Carefully transfer 50uL of cells to the ligation reactions tubes from Step 2. Use 100uL of cells for the uncut DNA control tube. Gently flick the tubes and incubate on ice for 20 minutes.
5. Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C. Do note shake. Immediately return the tubes to ice for 2 minutes.
6. Add 950uL room temperature SOC medium to the ligation reaction transformants at 900uL to the uncut DNA control tube. Incubate for 1.5 hours at 37C with shaking (approximately 150 rpm).
7. Plate 100uL of each transformation culture onto duplicate LB/ampicillin/IPTG/X-Gal plates. For the uncut DNA control, a 1:100 dilution with SOC is recommended.
8. Incubate plates overnight at 37C. Select white colonies.

Please note that we made minor modifications to this protocol:

Qiagen Plasmid Midi Prep Kit:

1. Resuspend completely the bacterial pellet in 4mL of Buffer P1 (ensure that RNase A has been added)
2. Add 4mL of buffer P2 (mix gently by inverting 4-6 times), incubate at room temperature for no more than 5 minutes
3. Add 4mL of chilled buffer P3 (mix by inverting 4-6 times), incubate on ice for 15 minutes (becomes cloudy and viscous)
4. Centrifuge at > 20,000 g for 30 minutes at 4C, remove supernatant containing the plasmid DNA promptly
5. Re-centrifuge supernatant > 20,000 g for 15 minutes at 4C, remove supernatant containing the plasmid DNA promptly
6. Equilibrate a QIAGEN-100 tip by applying 4mL of buffer QB and allow the column to empty by gravity flow (allow to drain completely)
7. Apply the supernatnat from step 8 to the QIAGEN-100 tip promptly
8. Wash the QIAGEN-100 tip with 2 x 10mL buffer QC
9. Elute DNA with 5mL buffer QF (collect eluate)
10. Precipitate DNA by adding 3.5mL(0.7 volumes) room-temp isopropanyl to the eluted DNA, mix and centrifuge immediately at 15,000 g for 30 minutes at 4C
11. Wash DNA with 2mL of room temperature 70% ethanol and centrifuge at 15,000g for 10 minutes (carefully decant without disturbing the pellet)
12. Air dry pellet and redissolve in appropriate volume of TE buffer

Please note that we made a minor modification to this protocol:

Making LB Media and plates:

LB (per litre):

Dissolve the following in 800 mL MilliQ water:

10 g tryptone

5 g yeast extract

10 g NaCl

Bring volume to 1 L and autoclave as indicated below (121C, 15 min).

LB agar: Add 7.5 g agar per 500 mL media in a 1 L flask. The agar will not dissolve, so just add it directly to the flask after pouring in the LB media. Cover the flask with foil and sterilize by autoclaving.

Antibiotic addition: Carbenicillin is more stable in media than ampicillin, and is used in plates to help prevent the growth of satellite colonies. Used at 100g/mL final concentration.

For LB cb100 plates, cool flask (500 mL LB agar) for about 30 min after autoclaving in a 50C water bath.

Add carbenicillin (0.5 mL of 100 mg/mL stock), swirl flask and pour plates (yield is 20-25 100 mm plates).

Flame out air bubbles. Dry plates in hood (or 1 day on the bench) and store in the fridge.

Making a GelRed stained agarose gel (for 1%)

Weigh out 1g agar and dissolve it in 100mL of TAE buffer (Tris-Acetate-EDTA buffer) in a conical flask and swirl. Put glad wrap to cover the flask and perforate a few times with something sharp.

Microwave the gel so that it melts completely. Let it cool on the benchtop or by running cool water on to the outside of the flask.

Once cooled, pour the liquid into the gel tray and put the combs into position.

Next add GelRed this is a chemical that intercalates DNA and makes it visible under UV light. The amount of GelRed to be added is as follows: of a XXmg/mL stock solution (that’s what most of the stuff around the lab is), add 1/XXXto your gel. For example, if we go back to our XXmL gel, then you would add XX°L of GelRed.

Allow the gel to harden (approximately 20 minutes) then take the combs out and cover with TAE buffer. The gel is now ready to be loaded with sample.

Heat Shock Transformation

1. Prepare LB agar plates and required antibiotics. Each transformation requires 2 plates.
2. Turn on water bath to exactly 42C. It takes at least 30 minutes to stabilize temperature.
3. Remove competent cells from -80C freezer and allow to thaw gently on ice for 5 to 10 minutes. Do not warm in hands as this can damage the cells.
4. Pipette 1uL plasmid (as long as concentration is >1ng/uL) into an autoclaved 1.5mL Eppendorf tube. (Negative control reaction to test for antibiotic solutions – use 1uL of ddH20 in place of plasmid. There should be no colony growth).
5. Add 15uL BL21(DE3) competent cells (expression cell line) to plasmid and flick gently to mix. Place left over competent cells back in -80C freezer.
6. Incubate reaction on ice for 30 minutes.
7. Heat shock for 40 seconds at exactly 42C. Do not shake.
8. Place on ice for 2 minutes.
9. Add 80uL SOC medium
10. Incubate 1 hour at 37C with shaking at 200 rpm
11. Spread 1 x 30uL and 1 x 70uL aliquots onto two separate LB agar plates with spreader + carbecillin antibiotic
12. Place plates in 37C incubator and incubate plates with agar on bottom for approximately 30 minutes to help dry
13. Turn plates over and incubate at 37C overnight
14. The plates can be stored and sealed with parafilm for less than 5 days only
15. Prepare glycerol stocks of cell lines and test for protein expression