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: