Team:UCL/Labbook/Week12

From 2013.igem.org

(Difference between revisions)

Revision as of 22:01, 4 October 2013

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, selective plates were spread, incubated at 37°C overnight.

Inoculations 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.

Tuesday 20th August

Plates taken from incubation:

Competent cells transformed with plasmid: Colony growth indicating cells have successfully taken up plasmid.

Negative control (only competent cells): Colony growth indicating likely problems with chloramphenicol.

Chloramphenicol will therefore be re-made.

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 -> storage at -20°C.

RFP+PSB1C3 -> 4x 200ul glycerols/a> -> glycerol storage at -20°C.

All 4 Falcons were minipreped. Subsequently ran gel showed no bands indicating no DNA presence.

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 chloramphenicol plates were made using supervisor Yanika Borg's chloramphenicol.

Meeting with Dr Darren Nesbeth:

The following Biobricks ordered were brought by our supervisor to be streaked onto the relevant antibiotic plates and inoculated in antibiotic+2mL LB. Incubate at 37°C overnight.

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, 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 made from the plates that displayed sufficient 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

@@ Line 65: / Line 65: @@
 </p>
 <p class="body_text">
-<b>Monday 19th August</b> - <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> transformation</a>. Colonies were expected to be red in colour, however this was not observed.
+<b>Monday 19th August</b>
+</p>
+<p class="body_text">
+<a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> streaked</a> plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a>. Colonies were expected to be red in colour, however this was not observed.
 </p>
@@ Line 86: / Line 90: @@
 </p>
 <p class="body_text">
-A single colony from each plate was then inoculated in LB in order to later prepare <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a>.
+A single colony from each plate was then inoculated in LB in order to later prepare <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerol stocks</a>.
- </p>
+</p>
 <p class="body_text">
-iGEM 2012 boxes were searched for <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>. <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Transformation</a> was carried out and then <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> selective plates</a> were spread, incubated o/n @37C.
+iGEM 2012 boxes were searched for <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>. <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Transformation</a> was carried out, <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> selective plates</a> were spread, incubated at 37°C overnight.
 </p>
 <p class="body_text">
-Innoculations of the following were carried out in 2mL LB:
+Inoculations of the following were carried out in 2mL LB:
 </p>
 <p class="body_text">
-·         IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker
+·IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker
 </p>
 <p class="body_text">
-·         RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker
+·RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker
 </p>
 <p class="body_text">
-^ 1.5mL miniprep, 0.5mL glycerol stock for following day (20th)
+.5mL miniprep, 0.5mL glycerol stock for following day.
-</p>
 </p>
+<p class="body_text">
+<b>Tuesday 20th August</b>
 </p>
 <p class="body_text">
-<b>Tuesday 20th August</b> - Plates taken from incubation:
+Plates taken from incubation:
 </p>
 <p class="body_text">
-Competent cells transformed with plasmid: Colony growth -> cells have successfully taken up plamid.
+Competent cells transformed with plasmid: Colony growth indicating cells have successfully taken up plasmid.
 </p>
 <p class="body_text">
-Negative control (only competent cells): Colony growth -> Most likely problems with <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a>.
+Negative control (only competent cells): Colony growth indicating likely problems with <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> chloramphenicol</a>.
 </p>
 <p class="body_text">
-Will therefore re-make <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> chloramphenicol</a>
+<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Chloramphenicol</a> will therefore be re-made.
 </p>
@@ Line 156: / Line 161: @@
 </p>
 <p class="body_text">
-IRRE+PC+RBS (<a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>) -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerols</a> -> glycerol box -20C mmp
+IRRE+PC+RBS (<a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a>) -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerols</a> -> storage at -20°C.
 </p>
 <p class="body_text">
-RFP+PSB1C3 -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerols/a> ->glycerol box -20C mmp
+RFP+PSB1C3 -> 4x 200ul <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> glycerols/a> -> glycerol storage at -20°C.
 </p>
 <p class="body_text">
-<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Minipreped</a> all 4 falcons and a gel was run -> no bands were seen indicating no DNA present.
+All 4 Falcons were <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> minipreped</a>. Subsequently ran gel showed no bands indicating no DNA presence.
 </p>
 <p class="body_text">
 <table>
@@ Line 260: / Line 265: @@
 </p>
 <p class="body_text">
-<b>Wednesday 21st August</b> - Nanodrop of pDNA
+<b>Wednesday 21st August</b>
+</p>
+<p class="body_text">
+Nanodrop of pDNA
 </p>
 <p class="body_text">
@@ Line 293: / Line 300: @@
 </p>
 <p class="body_text">
-x cmp plates were made using Yanika's chloramphenicol
+x chloramphenicol plates were made using supervisor Yanika Borg's chloramphenicol.
 </p>
 <p class="body_text">
-Meeting with Darren:
+Meeting with Dr Darren Nesbeth:
 </p>
 <p class="body_text">
-The following Biobricks ordered were brought by our supervisor to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto the relevant <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> antibiotic plates</a> and inoculated in antibiotic+2mL LB -> Incubate o/n @ 37C.
+The following Biobricks ordered were brought by our supervisor to be <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaked</a> onto the relevant <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> antibiotic plates</a> and inoculated in antibiotic+2mL LB. Incubate at 37°C overnight.
 </p>
 <p class="body_text">
@@ Line 347: / Line 353: @@
 </p>
 <p class="body_text">
-A <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> PCR</a> of Zeocin was performed and a gel was subsequently ran.
+A <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> PCR</a> of Zeocin was performed, a gel was subsequently ran.
+</p>
+<p class="body_text">
 PCR tube:
 </p>
@@ Line 369: / Line 377: @@
 </p>
 <p class="body_text">
-PCR was unsuccessful
+PCR was unsuccessful.
 </p>
 <p class="body_text">
-<b>Thursday 22nd August</b> -
+<b>Thursday 22nd August</b>
 </p>
 <p class="body_text">
-Results from Biobrick <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> streaking</a> and inoculation:
+Results from Biobrick <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> streaking</a> and inoculation:
 </p>
 <p class="body_text">
 <table>
@@ Line 441: / Line 449: @@
 </p>
 <p class="body_text">
-<a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> Glycerol stocks</a> were therefore made from the plates that displayed colony growth.
+<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Glycerol stocks</a> were made from the plates that displayed sufficient colony growth.
 </p>
 <p class="body_text">
-A second attempt at zeocin <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> PCR</a> was performed using Phusion DNA Polymerase
+A second attempt at zeocin <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> PCR</a> was performed using Phusion DNA Polymerase.
 </p>
 <p class="body_text">
@@ Line 465: / Line 473: @@
 - Negative control zec F, R
 </p>
 <p class="body_text">
 A gel was run with 8ul of each of the 6 reactions. PCR was successful.
 </p>
 <p class="body_text">
 Restriction digest of the following samples:
@@ Line 486: / Line 495: @@
 <p class="body_text">
 E - <a href="http://parts.igem.org/Part:BBa_J63009" target="_blank"> J63009/8</a>
 </p>
 <p class="body_text">
 <table>
@@ Line 534: / Line 543: @@
 <div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/8/89/Aug_21_UCLiGEM2013.jpg');height:488px;width:650px"></div>
 <p class="body_text">Nanodrop of samples:</p>
 </p>
 <p class="body_text">
 <table>
@@ Line 569: / Line 578: @@
 </tr>
 </table>
 </p>
 <p class="body_text">
 PCR Purification
@@ Line 587: / Line 596: @@
 <p class="body_text">
 Preparative digest of K812014 (pSB1C3)
 </p>
 <p class="body_text">
 <table>
@@ Line 620: / Line 629: @@
 </tr>
 </table>
 </p>
 <p class="body_text">
 ug sample = 44ul of 113.3 ng/ul
- </p>
+</p>
 <p class="body_text">
 CMV PCR
@@ Line 630: / Line 639: @@
 <p class="body_text">
 Primers used: 2s + 6FW, 2s + bbRE
 </p>
 <p class="body_text">
-<b>Friday 23rd August</b> -
+<b>Friday 23rd August</b>
 </p>
 <p class="body_text">
@@ Line 655: / Line 664: @@
 <p class="body_text">
 Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:
 </p>
 <p class="body_text">
 <table>
@@ Line 675: / Line 684: @@
 </tr>
 </table>
 </p>
 </div>
@@ Line 690: / Line 698: @@
 </p>
 <p class="body_text">
-<b>Monday 19th August</b> - Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL
+<b>Monday 19th August</b>
+Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL
 </p>
@@ Line 779: / Line 788: @@
 </p>
 <p class="body_text">
-<b>Tuesday 20th August</b> - Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml
+<b>Tuesday 20th August</b>
+</p>
+<p class="body_text">
+Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml
 </p>
 <p class="body_text">
@@ Line 861: / Line 872: @@
 </tr>
 </table>
+</p>
+<p class="body_text">
+<b>Wednesday 21st August</b>
 </p>
 <p class="body_text">
-<b>Wednesday 21st August</b> - Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml
+Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml
 </p>
 <p class="body_text">
 <b>Thursday 22nd August</b> - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml
 </p>
 </div>