Team:UCL/Labbook/Week12

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Lab Weeks

Week 12

Bacterial Lab

Monday 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)

Tuesday 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

Wednesday 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

Thursday 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

Friday 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

Monday 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

Tuesday 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

Wednesday 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

Thursday 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