Team:Carnegie Mellon/Week3

From 2013.igem.org

Monday, June 24 9:20 am started new culture with 1088, 1089, and 1090 in LB with maltose and Mg (in incubator on 2nd floor)
0.5 L LB/ amp agar
1 ml amp/ 1000 ml agar
Start new overnights from plates for VSC537, 1088, 1089, and 1090
Streaked XL10 Gold Ultracompetent cells
streak VSC537, 1088, 1089, and 1090
Need to start overnight cultures of KR and RFP

Tuesday, June 25th
Didn’t start RFP or KR cultures.
Streaked plates look excellent (single colonies)
Need to start overnights of the plates for tomorrow
Packaging extract started: 9:46 end: ~11:30
Need chloroform, IPTG/X-gal (from Cheryl)
Started new culture of VCS257 in LB w/ Mg and Maltose for afternoon infection ~12:30
Nanodrop results:
RFP: 5.8ng/µL 260/280=1.88 260/230=.04
KR: 26.4ng/µL 260/280=1.85 260/230=.1
Possible carbohydrate contamination indicated by low 260/230 ratios (possible agarose contamination?)

Start infection strain cultures at 12:15pm
Stored 100 10-3 10-4 10-5 dilutions at 4ºC
At 4pm, dilute, infect and plate
to top agar, add 15 µL IPTG and 25µL X-gal to 2.5mL top agar

Wednesday, June 26

Only one potential plaque was observed on 10^-3 plate made on Tuesday. Hopefully it is actually a plaque and not just an irregularity in the top agar.
VSC257 cells infected with phage (undiluted and 10^-1) ~1:00 pm.
inoculated 1 ml SM buffer with picked phage
200 μL of VSC257 cells from fridge with 10μL picked phage in SM
200 μL of VSC257 cells with 10μL of 10^-1 dilution of picked phage
200 μL of VSC257 cells with 10 μL of packaging control (iptg and x-gal added to top agar)
PCR RFP/KR
7x20µL reactions of RFP and KR with one tube of control for each (must repeat)
Induced RFP/KR cells at 1:45pm. No red color in either tube
2:50, no color in either tube
5:50, no color in either tube

PCR started again ending around 4:15
5-minute digests were set up for 7 minutes
Ligation is sitting at 12ºC overnight using Thermo T4 ligase

Thursday, June 27th
30 µL 1088 (lytic) cell line in 3 ml LB supplemented with maltose and Mg inoculated at 11am
@12pm checked OD600: 0.673
Pelleted, diluted in 4 ml 10mM Mg
Packaging will be complete at 12:50 (add SM and chloroform to stop reaction)
Added 500µL 1M Magnesium sulfate to packaging extracts by accident, added 500µL of SM to help correct. Extra magnesium shouldn’t cause problems with infection since the magnesium will be diluted in the cells and top agar.

Friday, June 28th

Phage plates from June 27th:
RFP plates look good-both clear and blue plaques on IPTG/X-gal plates
need to screen for phage which have KR in correct direction
approx. 20 clear plaques on 10^-2 dilution plate of RFP phage
1:35 pm started 1089 and 1090 for infection.
@2:19pm no observable increase in turbidity
@3:10pm.... still nothing :(
4:15pm...yay!
1089: OD600 of 0.366
1090: OD600 of 0.650
5:20: resuspend in 4 ml 10 mM Mg
Infected at 6pm with first 3 picked phages from 10^-2 dilution RFP plate from 6/27 in 1089 and 1090
Made more 10mM Mg
Make LB agar/pour plates/ autoclave 1ml tips
8x PCR of KillerRed ending 3:20pm.
NanoDrop results from yesterday’s PCR and digests:
KR digest: 54.6 ng/µL
260/280=2.01
260/230=.11
RFP digest: 72.1 ng/µL
260/280=1.83
260/230=.15
Possible lack of DNA for KR ligation although not likely. Will try to increase DNA concentration for subsequent digestions and ligations. In order to do this, the tube from yesterday’s undigested DNA (14µL left) and the 160µL from today’s PCR can be put onto a PCR purification column, cleaned and eluted in 30µL. Then digested and cleaned and then Nanodropped before the ligation step so achieve the .2µg requirement for the ligation.

118 ng/µL for digested clean EcoKR 1.7µL was used for the overnight ligation to achieve the ideal 200ng.

Saturday, June 28th Eric came in and began packaging ~9am
Kathy stopped the packaging and plated new KR phages

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