Team:NYU-AD/Notebook/Week1

Exterminator Coli

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Week 1\

15/07/2015

We started the miniprep using the instructions of QIAprep Spin Miniprep Kit:

• Add RNase A solution (200 ul of concentration mg/ul) to Buffer PI (20ml) and mix, store at 2-8˚C. This was stored in the fridge, 3rd from right. (We did not add the LyseBlue reagent to the Buffer PI )
• Centrifuge the overnight culture (1ml in each tube) of 8200rpm for 3mins at room temperature
• Pour out the LB, so that the pellet remains, then resuspend the pellet in 250 ug of Buffer solution. Then transfer to a microcentrifuge tube.
• Add the 250ug of P2, mix thoroughly. Do not allow to proceed for more than 5 mins.
• Add 350ug of Buffer N3 , mix immediately, invert 4-6 times.
• Centrifuge for 13,000 rpm for 10mins in the centrifuge
• Apply the supernatant, centrifuge for 30-60secs, discard flow through
• Add 500ul pf PB buffer to wash and centrifuge for 30-60seconds. This was an optional step that was done in our process
• Wash QIAprep spin column, add 750ul Buffer PE. Centrifuge for 30-60seconds. (We prepared glycerol stock of liquid culture, with equal parts of glycerol and cell culture of 500ul. This was stored in the -8˚C fridge.)
• Centrifuge for 1 min to remove residual wash buffer.
• Place QIAprep column in a clean eppendorf tube. Add 50ul of water ( not the Buffer EB), let it stand for 1 min, then centrifuge for 1 minute.

The DNA was stored in the fridge at 4 ˚C overnight.

Note: We used deionized water for step 10, so we are not sure if it is DNA free and RNA free. So we have to see if there is any degradation tomorrow.

16/07/2015

We performed a Nano drop to check the quality of DNA. We pipetted 1ul of DNA into the machine.

Results

-tr1, tr2, BBA006, BBA700 are of poor quality so we will grow and miniprep them again. With picking a colony, we take 250ul of LB then incubate for hours, then plate.

1. Use the culture from 12/7/15. (iGEM 2015 plates were from 13/07)
2. 250ul of LB Broth is added to each of the picked colonies and they go in the shaking incubator for several hours ( 2-3 hours), shaking at 180rpm. * colonies were picked from the plate of 006 and 700 cells, as well as from original agar stabs from iGEM.
3. Re-doing the miniprep for the rest of the tubes and we used the rest of the liquid culture to get a better result in the Nanodrop.
4. Follow QiaPrep Spin Miniprep Kit protocol, but this time we added LyseBlue to the mixture, perhaps there was too little of solution because it did not turn blue but we could see the precipitate (i.e. cloudy, but colourless solution)
5. Skipped Step 7 of the protocol as it was optional, i.e. we did not add Buffer PB.

* Instead of deionised water, we added Buffer EB to elute

RESULTS OF 2nd Nano drop:

Results Of 2nd Nano Drop

Tube	Nucleic acid ng/ul	A260	A280	260/280	260/360
PR1	99.3	1.986	1.052	1.89	1.77
PR2	75.6	1.511	0.787	1.92	1.64
TR1	48.6	0.973	0.491	1.98	1.67
TR2	63.1	1.262	0.653	1.93	1.82
PR5	37.5	0.749	0.401	1.87	0.98
BBA006	49.0	0.981	0.510	1.92	1.46

15/07/2015

• The plated cells from liquid culture (16/07/15 ) that were inoculated from 250ul liquid cultures show good growth. These plates were raised from agar stabs shipped from iGEM.
• We picked single colonies from each plate and inoculated in LB and Chloroamphenicol liquid medium. Each colony picked was placed in 10ml of L.B. liquid medium in each tube. The inoculation occurred under sterile conditions i.e. under the fume hood with a Bunsen burner.
• The plates were stored in 4˚C refrigerator , covered with Parafilm tape.
• The liquid culture tubes were incubated in the shaking incubator at 1415h.
• The incubation conditions: 37˚C, horizontal shaking at 180rpm.

18/07/2015

• The liquid culture inoculated on 17/07 showed good growth based on optical density (visual inspection). Sediment observed in each tube in addition to turbidity in liquid media, indicative of high bacterial concentration.
• Liquid culture tubes were removed from shaking incubator, sealed with Parafilm and placed in 4˚C refrigerator at 1335h.
• Agar plates from transformations carried out on competent cells from Kenan remain in the incubator. Both promoter plates show moderate growth while terminator plates remain clear with no visible growth.