Team:UCL/Labbook/Week12

Bacterial Lab

19th August - pSB1C3 streaked plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful transformation. Colonies were expected to be red in colour, however this was not observed.

Plate (competent cell vial)	Colony Count
10	50+
20	50+

A single colony from each plate was then inoculated in LB in order to later prepare glycerol stocks.

iGEM 2012 boxes were searched for pSB1C3. Transformation was carried out and then selective plates were spread, incubated o/n @37C.

Innoculations of the following were carried out in 2mL LB:

· IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker

· RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker

^ 1.5mL miniprep, 0.5mL glycerol stock for following day (20th)

20th August - Plates taken from incubation:

Competent cells transformed with plasmid: Colony growth -> cells have successfully taken up plamid.

Negative control (only competent cells): Colony growth -> Most likely problems with Chloramphenicol.

Will therefore re-make chloramphenicol

Plate (pSB1C3+)	Colony Count
NUC	5
LAC	20
LAC	15
LAC2	10
CURL1	30
Control	50

take from incu-shaker:

IRRE+PC+RBS ( pSB1C3) -> 4x 200ul glycerols -> glycerol box -20C mmp

RFP+PSB1C3 -> 4x 200ul glycerols/a> ->glycerol box -20C mmp

Minipreped all 4 falcons and a gel was run -> no bands were seen indicating no DNA present.

Item (ul)	IrreAcut	IrreAuc	IrreBcut	IrreBuc	RFPcut	RFPuc	Uncut	Uncut
pSB1C3	5	5	5	5	5	5	5	5
EcoR1	1	0	1	0	1	0	1	0
Pst1	1	0	1	0	1	0	1	0
Buffer 3	1	0	1	0	1	0	1	0
BSA	0.5	0	0.5	0	0.5	0	0.5	0
dH20	1.5	5	1.5	5	1.5	5	1.5	5
Total	10	10	10	10	10	10	10	10

21st August - Nanodrop of pDNA

Sample	Concentration (ng/ul)	260/280
Irre A pDNA	-33.0	1.74
Irre b pDNA	123.0	0.94
GFP pDNA	-52.1	1.63
LB pDNA	-28.4	1.34

10x cmp plates were made using Yanika's chloramphenicol

Meeting with Darren:

The following Biobricks ordered were brought by our supervisor to be streaked onto the relevant antibiotic plates and inoculated in antibiotic+2mL LB -> Incubate o/n @ 37C.

Biobrick	Plate (+ND)	Inoculation (+ 2mL LB)
J63008	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP
K105028	1X AMP	2 mL AMP
I712004	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP
K105030	1X AMP	2 mL AMP
J63008/9	1X AMP	2 mL AMP
K105027	1X AMP	2 mL AMP
K812014	1X AMP, 1X CMP	2 mL AMP + 2 mL CMP

A PCR of Zeocin was performed and a gel was subsequently ran. PCR tube:

1 - PCR zec bb F, R, 2ul template

2 - PCR zec bb F, R, 1ul template

3 - PCR zec F, R, 2ul template

4 - PCR zec F, R, 1ul template

5 - PCR negative control zec bb F, R,

6 - PCR negative control zec F, R

PCR was unsuccessful

22nd August -

Results from Biobrick streaking and inoculation:

Biobrick	Positive Control (ND)	Cmp plate	Amp plate	Falcon
J63008	Growth	Growth	No Growth	No Growth
K105028	Growth		Growth	Growth
K105027	Growth		Growth	Growth
K105030	Growth		Growth	Growth
K812014	Growth	Growth		Growth
J63008/9	Growth		Growth	Growth
I712004	No Growth	No Growth	No Growth	No Growth

Glycerol stocks were therefore made from the plates that displayed colony growth.

A second attempt at zeocin PCR was performed using Phusion DNA Polymerase

1 - Zec bb F, R 2ul

2 - Zec bb F, R 1ul

3 - Zec F, R 2ul

4 - Zec F, R 1ul

5 - Negative control zec bb F, R

6 - Negative control zec F, R

A gel was run with 8ul of each of the 6 reactions. PCR was successful.

Restriction digest of the following samples:

A - K105028

B - K105027

C - K105030

D - K812014

E - J63009/8

	Double Digest	Uncut
Sample	5	5
EcoR1	1	0
Pst1	1	0
Buffer 3	1	0
BSA	0.5	0
dH2O	1.5	5
Total	10	10

Nanodrop of samples:

Sample	260/280	ng/ul
K105028	2.12	39.0
K105027	1.99	47.1
K105030	2.07	48.8
K812014	1.96	113.3
J63008/9	1.95	59.7

PCR Purification

1 - zeo bb F, R 2ul template

2 - zeo bb F, R 1ul template

^ stored in iGEM 2013 box

Preparative digest of K812014 (pSB1C3)

Sample	5ug
E	7
P	7
B3	10
BSA	2
dH2O	30
Total	100

5ug sample = 44ul of 113.3 ng/ul

CMV PCR

Primers used: 2s + 6FW, 2s + bbRE

23rd August -

Gel was ran with (lane):

(3) PCR purified zeo bb FR + 2ul template

(4) PCR purified zeo bb FR + 1ul template

(6) bwf + bb RE + 1ul template

(7) bwf + bb RE + 2ul template

(8) bwf + bb RE

Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:

Sample	260/280	ng/ul
PCR purified zeo bb FR + 2ul template	1.66	55.6
PCR purified zeo bb FR + 1ul template	1.82	17.2

Mammalian Lab

19th August - Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	100	Over confluent	Moderate	100	Over confluent	moderate
50	50	Moderate swelling and death	Many	80	Moderate swelling and death	Many
100	20	severe swelling, death	Many	20	severe swelling, death	many
250	0	-	many	0	-	many
500	0	-	many	0	-	many
1000	0	-	many	0	-	many

Split stock HeLa cells

20th August - Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	100	Over confluent and death	Moderate	90	Over confluent	moderate
50	80	Over confluent, swelling and death	Many	20	Moderate swelling and death	Many
100	20	severe swelling and death	Many	0	severe swelling, death	many
250	0	-	many	0	-	many
500	0	-	many	0	-	many
1000	0	-	many	0	-	many

21st August - Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml

22nd August - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml