Team:UCL/MammalianLabs

June

Week 1-3

No lab work

Week 4

26th June - We are given safety training in the Advanced Centre for Biochemical Engineering in all relevant laboratories, as well as general procedures in case of emergency.

July

Week 5-6

No lab work

Week 7

Bacterial Lab

15th July - The team is introduced to the laboratories which will be used during the summer for both bacterial and mammalian experiments.

16th July - 5X M9 salts, minimal agar, 1.4% molten agar solution and 0.1M CaCl2/15% glycerol were prepared for the generation of competent cells. Minimal agar plates were poured and streaked with W3110 Escherichia coli cells and left overnight to incubate at 37C.

17th July - Very little colony growth was observed from W3110 ''E.coli'' streaked plates. Plates were therefore left to incubate for a further 17 hours.

18th July - Sufficient colony growth allowed for the selection of a single colony from each plate. This was then inoculated in 5ul LB media + 100ul 1M MgSO4 and left to incu-shake overnight at 37C.

19th July - Cultures re-suspended in new LB media and 100µl aliquots placed into individual eppendorf tubes for placement at -80C.

Mammalian Lab

17th July - Mammalian cell culture and maintenance training by Mrs. Ludmilla Ruban. Passaged primary MEF (mouse embryonic fibroblast) cells. Passage 3.

18th July - MEF passage 4

19th July - MEF passage 5

Week 8

Bacterial Lab

22nd July - Transformation of our competent cells with plasmid YB3110 was carried out.

23rd July - No colony growth was observed on Ampicillin plates indicating no plasmid uptake. transformation was repeated with YB3110.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

24th July - No colony growth from main experiment indicating no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

25th July - No colony growth and hence no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

26th July - Once again our cells were unsuccessful in taking up the YB3110 plasmid. We concluded that our batch of competent cells were not competent and so these will not be used in any further experiments. A new batch of competent cells are to be generated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

Mammalian Lab

22nd July - MEF passage 6. Training ends.

Week 9

Bacterial Lab

29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.

30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

31st July - A new stock of competent cells were generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.

Ampicillin was produced and stored. Following miniprep of pSecTag2A a gel was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A]. 50X TAE diluted to 1X.

Item	Volume (ul)
DNA pSecTag2A	5
HindIII	1
Buffer	1
BSA	0.5
dH20	2.5
Total	10

Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.

August

1st August - Results from newly generated competent cells transformed with pSecTag2A:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	10

This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the transformation protocol from yesterday was repeated to see whether the ampicilin was working -> plates left for incubation 37C o/n.

Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.

2nd August - Plates displayed significant colony growth on amp plates indicating successful transformation. Negative control however displayed slight colony growth - ampicillin not working effectively, new ampicillin was therefore prepared. End of week inventory was recorded and stocks were topped up.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

Mammalian Lab

29th July - Thawed and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu

30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend

Week 10

Bacterial Lab

5th August - The new batch of ampicillin was tested via transformation of competent cells with pSecTag2A. Plates were spread and incubated 37C o/n.

6th August - Results from yesterday’s transformation:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

This indicates that there is still an issue with the ampicillin, possibly a problem with the stock powder used. A final test with both old and new ampicillin was carried out and compared without plasmid insertion.

7th August -

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 Old Ampicillin	Yes	Yes	100+
2 New Ampicillin v2	No	Yes	100+
3 Positive Control	Yes	No	15

Results indicated that the ampicillin source may not have been fully functional, therefore a new source of amp powder was located and amp was remade.

8th August - Preparation of 4x Amp plates and 4x no drug plates for storage in the fridge.

9th August - Meeting with Darren Nesbeth, requirements for upcoming weeks: create glycerol stocks of both pSecTag2A and pSB1C3, purify and extract pure DNA for pSecTag2A and pSB1C3.

Mammalian Lab

6th August - Checked HeLa cells. Cells are growing slow, left to grow for a few more days

8th August - HeLa cells are about 30% confluent. Changed media.

Week 11

Bacterial Lab

12th August - Chloramphenicol was produced and stored at -20C. 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 37C o/n.

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 also 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. Amp & Cmp were remade and pSB1C3 was located in the 2012 distribution kit. Amp & Cmp were both tested by producing 1x positive and 1x negative plate for each antibiotic streaked with W3110 cells ->left to incubate o/n at 37C. 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.

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]

[link to enzyme conditions]

The pSB1C3 glyc stock (from HQ) was from the 2012 iGEM box in MMP -20C

Told to use a ratio of 3:1 inoculum:80% glycerol when making glyc 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 cmp and ND plates again.

pSecTag2A

50ul of 2013 pSecTag2A glyc stock to inoculate 10mL LB Amp in a 50mL Falcon. Grow overnight and the next day generate 15x glyc stocks sing 1.5mL eppendorfs -> store in MMP -20C.

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 Amp glycerol stock and 4x eppendorfs of pSB1C3 ND glycerol stock was prepared. pSB1C3 LB ND was streaked onto two plates (1x Cmp, 1xND) ->incubate o/n @37C 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

[insert image of gel]

iRRE+PC+RBS and PC+RBS (containing pSB1C3, taken from 2012 iGEM boxes) were plated onto cmp plates -> incubated o/n @37C

Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml cmp -> incushaker o/n

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 o/n @30C over the weekend.

Mammalian Lab

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).

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

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.

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

17th August - Passaged HeLa cells

Week 12

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

Week 13

Bacterial Lab

27th August -

Purify zeocin CMV from zeo FB primers

Zeocin and CMV purification

Tube:

1 - CMV 1ul template bb F, R

2 - CMV 2ul template bb F, R

3 - Zeocin 2ul template zecF, R

4 - Zeocin 1ul template zecF, R

5 - Zeocin 2ul template zecbbF, R

6 - Zeocin 1ul template zecbbF, R

28th August - 3x glycerol stocks of K812014 were prepared from inoculum. 1.5mL of culture was used for miniprep.

Nanodrop of Zec and CMV was recorded:

	260/280	ng/ul
ZEC	1.68	21.1
CMV	1.96	82.1

A gel was run on 100ul of prep digest K812014 + 20ul dye. 2000bp was scooped in gel extraction.

Chloramphenicol was tested at x2 and x4 concentration with 50ul cells spread on each plate, results from the next day still showed significant colony growth (100+).

29th August - Nanodrop of pSB1C3 purified from gel extract, 260/280: -25/21, ng/ul: 13.5

5ul was run on a gel, however extract of pSB1C3 was unsuccessful as no bands were visible.

PCR:

3 PCR reactions of zeocin using zec BB F, R primers were performed. First reaction: 1 ul pSecTag2A template, second 2 ul pSecTag2A and third reaction - negative control - no template.

3 PCR reactions for cmv promoter were prepared using the same variation of template volume as above. These were tested the next day and were successful. Gel photo (30/08).

Prep digest+gel+extract+purify+nanodrop+5ul gel of zeocin and CMV were carried out

Prep digest:

Sample (ul)	Zeo	CMV	Cyc100	Cyc100	Cyc70	Cyc70	Cyc28	Cyc28
V. Sample	48	45	17.5	17.5	23	23	23.5	23.5
B3	10	10	5	5	5	5	5	5
E	7	7	5		5		5
P	7	7	5	5	5	5	5	5
X				5		5
BSA	2	2	1	1	1	1	1	1
H2O	26	29	16.5	16.5	11	11	10.5	10.5
Total	100	100	50	50	50	50	50	50

Glycerol stocks (x15) of J63009, K105027, K105028 and K105030 were made.

Additionally, nanodrops were recorded:

Sample	260/280	Absorbance	ng/ul
J63009	2.10	0.774	88.7
K105030	209.00	0.653	60
K105028	2.14	0.408	50
K105029	2.18	0.585	77.0
K218014	2.12	0.946	104.9

A prep digest was conducted on K812014 to extract pSB1C3.

Chloramphenicol was tested again but this time with x8 cmp (80ul cmp to 10mL LB agar). Overgrad, undergrad and Yanika chloramphenicol stocks were used and 50ul competent cells were spread -> incubate o/n @37C.

Zec and CMV nanodrops were recorded:

Sample	260/280	Absorbance	ng/ul
ZEC	1.87	9.538	2.5
CMV	2.06	11.048	25.4

30th August

Chloramphenicol test - plate colony growth observation and count: all 3 plates prepared using the chloramphenicol from postgrads, us and Yanika had 30+ very small colonies.

After overnight incubation, from each of the the 10 ml inoculations of K812014 (I), pSecTag2A and second (different) sample of K812014 (II), 2.5 ml were minipreped while the rest of 7.5 ml from each was used to make glycerol stocks.

Nanodrop of these minipreps:

Sample	ng/ul	260/280	260/230	Absorbance
K812014 (I)	117.8	2.10	2.49	0.944
pSecTag2A	35.9	1.81	1.44	0.496
K812014 (II)	10.7	1.71	2.55	0.084

Prep digest of K812014 (I) - miniprep sample prepared by Andy. Contents of reaction:

Sample K812014 (I)	30 ul
EcoRI (E)	5 ul
PstI (P)	5 ul
BSA	1 ul
Buffer 3	5 ul
Total	4 ul
Total Volume of Reaction	50 ul

A gel was run with the E, P cut K812014 (Gel photo?) and the corresponding band of liniarised pSB1C3 (backbone of K812014) was extracted for purification.

Nanodrop of the purified pSB1C3 DNA showed a concentration of 8.4 ng/ul (260/290 = 2.91; 260/230 = 0.02, Absorbance = 7.379) while the nanodrop of the pSB1C3 DNA offered by the postgrads showed 13.01 ng/ul (260/280=1.34).

PCR:

8 new reactions were prepared for zeocin - same primers bb F,R as used previously.

A checking (analytical) gel was performed revealing no bands for reaction tubes 6 and 7 (each of these with 1 ul template)- PCR unsuccessful.

These amplified zeocin together with the 2 reaction tubes of amplified zeocin f aacmv from a day before and a were loaded onto the gel and the bands corresponding to 2 kb were extracted for purification.

31 August -

Gel extraction purification of zeocin and cmv. We are expecting that through this procedure the Phusion polymerase, DMSO, buffer, template DNA will be washed away, thus achieving purification of zeocin and cmv DNA (which are to be digested hence, prepared for ligation in the pSB1C3 backbone).

PCR:

Zeocin was amplified again, in 9 tubes (same reaction) -> a total volume of 50 ul x 9 = 450 ul.

Prep digest of zeocin and cmv straight after PCR.

Components	Prep digest Cmv	Prep digest Zeocin
DNA sample	60 ul	60 ul
EcoRI	7 ul	7 ul
PstI	7 ul	7 ul
Buffer 3	10 ul	10 ul
BSA	2 ul	2 ul
Water	14 ul	14 ul
Total	100 ul	100 ul

These digests were used for LIGATION 1

LIGATION 1: using

pSB1C3 digested and purified	10 ng/ul
Zeocin digested and purified	55 ng/ul
Cmv digested and purified	20 ng/ul

Ligation of zeocin to pSB1C3:

Zeocin (55 ng/ul)	3 to 1	6 to 1
Water (ul)	2.1	0
Quick ligase buffer (ul)	10 ul	10 ul
Backbone (ul)	5 ul	5 ul
Insert (ul)	2.7 ul	5.5 ul
Ligase (ul)	1 ul	1 ul