Team:UCL/Labbook/Week13

Bacterial Lab

Tuesday 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

Wednesday 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+).

Thursday 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

Friday 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.

Saturday 31st 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	10
Backbone (ul)	5	5
Insert (ul)	2.7	5.5
Ligase (ul)	1 ul	1 ul
Total (ul)	21	21.5

Ligation of cmv to pSB1C3:

(20 ng/ul)	3 to 1	6 to 1
Water (ul)	2.4	0
Quick ligase buffer (ul)	10	10
Backbone (ul)	5	5
Insert (ul)	2.6	5.3
Ligase (ul)	1 ul	1 ul
Total (ul)	21	21.3

Controls used:

Controls	1 check no circular backbone	2 check no digestion process	3 control (uncut backbone)
Water (ul)	5	6	9
Quick ligase buffer (ul)	10	10	10
Backbone (ul)	5	5	2 of uncut backbone
Insert (ul)	0	0	0
Ligase (ul)	1	0	0
Total (ul)	21	21	21

5 ul of each of each ligation reaction were used to transform our home-made competent cells (4 vials - all ligations apart from the 3 controls) as well as Yanika’s top 10 cells (7 vials - all ligations).

T10 - Top 10 cells

HM - home made competent cells

	T10	T10	T10	T10	T10	T10	T10	HM	HM	HM	HM
Cell vial no.	1	2	3	4	5	6	7	28	45	51	52
Vol. ligation plated (ul)	90 & 10	90 & 10	100	100	100	90 & 10	90 & 10	100	90 & 10	100	100
Ligation containing	zeo 3:1	zeo 6:1	control 1	control 2	control 3	zeo 3:1	zeo 6:1	zeo 3:1	zeo 6:1	zeo 3:1	zeo 6:1
Cell counts	0	0	0	0	0	0	0	15	0 & 10*	15	15

* 0 colonies in plate with 10 ul inoculum spread and 10 colonies for the 90 ul inoculum spread.

After the transformation protocol we spread the transformed cells on 4x cmp (?) agar plates using both 90 ul and 10 ul cell inoculum in the case of cell vials 1,2,6,7 (Yanika’s) and 45 (ours).

We picked 5 colonies from each plated cell vials 28, 45, 51 and 52 (2 ml and 2 ul cmp) and incubated for 10 hours. These inoculations were then used to make glycerol stocks and minipreps.

Gel extraction purification

The 9 tubes of amplified zeocin performed were loaded on a gel and the 2 kb bands were subsequently extracted (6.2 g of gel). This zeocin DNA was purified using gel extraction purification kit and 3 tubes of each 60 ul purified zeocin resulted (labelling zeo g.e.p.).

Miniprep of K812014 cell-containing-biobrick: in order to make stocks of pSB1C3, the backbone into which this biobrick is inserted.

Nanodrop of purified zeocin (from above):

Zeo g.e.p. tube	ng/ul	260/280
1	36.1	1.95
2	38.9	1.90
3	42.9	1.90

Nanodrop of miniprep of K812014: 197 ng/ul (260/280 = 1.94)

Sunday 1st September

The 20 inoculations were retrieved from the incu-shaker. All falcons showed growth apart from one inoculation from cell vial 28 (ligation 3:1 zeo).

1.5 ml of each tube was used for miniprep while the other 0.5 ml was used to make glycerol stocks.

Prep digest of K812014 (miniprep prepared on the 31 of August)

Component	Volume added (ul)
K812014 DNA	25
EcoRI	5
PstI	5
Buffer 3	5
BSA	1
dH2O	9
Total	50

Nanodrop result of the miniprep of transformations:

Cell vial/colony no.	ng/ul	260/280
28/1	84.2	1.65
28/2	58.5	1.77
28/3	49.6	1.83
28/4	14.6	1.97
45/1	130.9	1.72
45/2	18.7	2.34
45/3	49.6	1.73
45/4	111.3	1.64
45/5	23.9	1.96
51/1	69.5	1.70
51/2	60.9	1.78
51/3	41.2	1.85
51/4	50.9	1.79
51/5	23.9	1.96
52/1	40.4	1.83
52/2	40.2	1.87
52/3	78.1	1.94
52/4	127.2	1.62
52/5	16.5	2.11

Analytical digest of the above minipreps - incubated at 37 degrees C for 1 hour

Volume (ul)	Single Digest (PstI)	Double Digest (EcoRI + PstI)
Miniprep	5	5
EcoRI	0	1
PstI	1	1
Buffer 3	1	1
BSA	0.5	0.5
dH2O	2.5	1.5
Total	10	10

These digests were loaded into a gel in the following order: single digest, double digest, uncut. The result of this gel was negative, there were no bands for the loaded DNAs (the 1 kb DNA ladder was on the gel), the procedures of achieving recombinant zeocin and cmv plasmid were unsuccessful.

We suspect that the water was contaminated with nuclease.

Mammalian Lab

Saturday 31st August - HeLa cells confluency: 100% and 40%. Split 100% confluency dish in 1:4, 40% confluency dish in 1:2