Team:UCL/Labbook/Week11

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Latest revision as of 23:53, 4 October 2013

Lab Weeks

Week 11

Bacterial Lab

Monday 12th August

Chloramphenicol was produced and stored at -20°C. In order to produce glycerol stocks, pSecTag2A and pSB1C3 were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37°C overnight.

Tuesday 13th August

Results from the following plates:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
pSecTag2A Cells	Yes	Yes	100+
W3110 Cells Amp	Yes	No	0
pSecTag +ve control	No	Yes	100+
PSB1C3 Cells	Yes	Yes	0
W3110 Cells Chlor	Yes	No	25
PSB1C3 +ve control	No	Yes	0

This indicated that the pSecTag2A cells are acceptable to use for glycerol stock generation and for plasmid purification. Miniprep was performed on the two incubated Falcon tubes from yesterday.

The results concerning pSB1C3 indicated that the chloramphenicol did not work, and the glycerol stock is dead. Therefore a new glycerol stock was sought after and chloramphenicol was remade.

Ampicillin & chloramphenicol were remade and pSB1C3 was located in the iGEM 2012 distribution kit. Ampicillin & chloramphenicol were both tested by producing 1x positive and 1x negative plate for each antibiotic streaked with W3110 cells. Left to incubate at 37°C overnight.

Glycerol stocks of pSecTag2A was grown, and a miniprep was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.

Wednesday 14th August

Two analytical digests of pSecTag2A with EcoR1-HF and Dpn1 were carried out.

Item A	Volume A (ul)	Item B	Volume B (ul)
pSecTag2A	5	pSecTag2A	5
EcoR1-HF	1	Dpn1	1
Buffer 4	1	Buffer 4	1
BSA	0.5	BSA	0.5
dH20	2.5	dH20	2.5
Total	10	Total	10

[insert image of gel]

Told by supervisor Dr Darren Nesbeth to use a ratio of 3:1 inoculum:80% glycerol when making glycerol stocks.

Darren's instructions:

pSB1C3

Transfer entire glycerol tube contents to 10mL LB ND in a 50mL Falcon & measure OD. Place in 37C shakee overnight. Measure OD next morning. If there is growth in the inoculum use a loop to streak onto chloramphenicol and ND plates again.

pSecTag2A

50ul of 2013 pSecTag2A glycerol stock to inoculate 10mL LB ampicillin in a 50mL Falcon. Grow overnight then generate 15x glycerol stocks in 1.5mL eppendorfs. Store at -20°C.

Thursday 15th August

OD results for pSB1C3 LB ND inoculum

Plasmid	OD Before	OD After
PSB1C3 (LB ND)	0.052	0.5

15x eppendorfs of pSecTag2A ampicillin glycerol stock and 4x eppendorfs of pSB1C3 ND glycerol stock was prepared.

pSB1C3 LB ND was streaked onto two plates (1x Chloramphenicol, 1xND). Incubated at 37°C overnight. Remaining 2ml of pSB1C3 LB ND (from falcon) is used to purify and conduct an analytical digest (16th August).

Restriction digest of pSecTag2A:

ul	EcoR1 single digest	Spe1 single digest	Double digest	Uncut
pSecTag2A	5	5	5	5
EcoR1	1	0	1	0
Spe1	0	1	1	0
BSA	0.5	0.5	0.5	0.5
Buffer 4	1	1	1	1
dH20	2.5	2.5	1.5	3.5
Total	10	10	10	10

iRRE+PC+RBS and PC+RBS (containing pSB1C3, taken from 2012 iGEM boxes) were plated onto cmp plates. Incubated at 37°C overnight.

Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml chloramphenicol. Incushaker at 37°C overnight.

Friday 16th August

Falcons were retrieved:

RESULTS

Inoculum:

Falcon contents (+LB+Amp) ul	Colony growth	Absorbance
IRRE+PC+RBS (15ul)	Yes	0.8
IRRE+PC+RBS (5ul)	Yes	0.7
PC+RBS (15ul)/td>	No	0.06
PC+RBS (5ul)	No	0.08

Plates

Plate contents (+LB+Amp) ul	Colony growth	Absorbance
IRRE+PC+RBS	Yes	100+
IRRE+PC+RBS	No	100+
PC+RBS/td>	Yes	0
PC+RBS	No	20

The PC+RBS plates & falcons were discarded

IRRE+PC+RBS (15ul inoculum) -> underwent miniprep

IRRE+PC+RBS (5ul inoculum) -> 4x glycerols were made: 500ul culture + 166ul 80% glycerol, stored in MMP -20C iGEM 2013 box.

pSB1C3 Gel sd: EcoR1 & Pst1 + dd

Item (ul)	EcoR1	Pst1	Double digest	Uncut
pSB1C3	5	5	5	5
EcoR1	1	0	1	0
Pst1	0	1	1	0
Buffer 3	1	1	1	1
BSA	0.5	0.5	0.5	0.5
dH20	2.5	2.5	1.5	4.5
Total	10	10	10	10

3ul loading dye to each solution

Lanes:

2 - Hyperladder, 4 - EcoRI, 5 - PstI, 6 - dd, 7 - Uncut

Results from gel: 1kb DNA ladder showed on gel. No bands for EcoR1, Pst1, DD and Uncut plasmid were seen - indicating that no DNA was present -> possibly due to problems with the miniprep.

Therefore from the iGEM 2012 plate 5 (distribution kit) well 23.O, DNA containing BBa_j04450 (colonies are clearly red in colour - RFP) in pSB1C3 was located. The dried DNA was resuspended in 10ul dH2O, left to sit for 5 minutes, then transformed into competent cells -> streaked onto plates and left to incubate at 30°C over the weekend.

Mammalian Lab

Monday 12th August

HeLa cells have confluency of about 60%. Passaged the HeLa cells and ‘backup’ HeLa cells. We set up two 6-well plates at 0.25 x 10^6 cells/ml in preparation for Zeocin kill curve. Cell count involved (0.45 cells/ml).

Tuesday 13th August

Prepared media with various concentrations of zeocin for each of 6 wells.

Concentration of zeocin (µg/ml)	Volume of zeocin (ml)	Volume of DMEM + 10% FBS + 2 mM L-Glu (ml)
0	0	30.0
50	15	30.0
100	30	30.0
250	75	29.9
500	150	29.9
1000	300	29.7

T75 flasks: - 90% confluency T25 flasks - 30% confluency 6-well plates - 30% confluency

Wednesday 14th August

T25 flasks 90% confluency

Disc 1 Disc 2

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Comment	Confluency (%)	Cell Appearance	Comment
0	65	Healthy	Healthy, few swell	65	Healthy	minimal floaters
50	40	Healthy, few swell	few floaters	65	occasional swell	moderate floaters
100	40	Half/moderate swell	Moderate floaters, minor infection	50	40% swelling	minimal floaters
250	25	most/moderate swell	many floaters	60	moderate/severe swelling	many floaters
500	40	most clumps dead/ swell	many floaters, possibly infection	65	severe swelling all over	large number of floaters
1000	45	very severe death/ swell	many floaters, minor infection	60	severe swelling all over	moderate number of floaters

The two T25 flasks with revived HeLa have confluency of about 90%; T75 flask with ‘backup’ cells 70% confluent. The T25 flasks are discarded.

Friday 16th August

		Disc 1			Disc 2
Concentration of zeocin (µg/ml)	Confluency (%)	Cell Appearance	Floaters	Confluency (%)	Cell Appearance	Floaters
0	90	Healthy	Minimal	95	Healthy	minimal
50	80	minor swell	moderate	90	minor swell	moderate
100	65	Minor swell, minor death	Many	75	Minor swell, minor death	moderate
250	50	severe swelling, death	many	50	severe swelling, death	many
500	60	severe swelling, death	many	50	severe swelling, death	many
1000	40	severe swelling, death	many	50	severe swelling, death	many

Split and passaged stock HeLa into 2 flasks

Saturday 17th August

Passaged HeLa cells

@@ Line 69: / Line 69: @@
 <p class="body_text">
 <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Chloramphenicol</a> was produced and stored at -20°C.
-In order to produce <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a>, pSecTag2A and <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37C o/n.
+In order to produce <a href="https://2013.igem.org/Team:UCL/Project/Protocols" target="_blank"> glycerol stocks</a>, pSecTag2A and <a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3" target="_blank"> pSB1C3</a> were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37°C overnight.
 </p>
 <p class="body_text">