Team:UCL/Labbook/Week14

Bacterial Lab

Monday 2nd September

We re-autoclaved water due to our suspicion it was nuclease contaminated. New inoculations (in 3 ml LB) of the transformations were prepared: 5 using cmv ligation and 5 using the zeocin ligation. These were left over night into the incushaker.

Tuesday 3rd September

The falcons were removed from the incu-shaker. Glycerol stocks were prepared using 0.5 ml while the rest was kept for minipreping.

Preparative digest of zeocin and K812014 plasmid

Components (ul)	Prep digest of Zeocin	Prep digest of K812014
Zeo/K812014	80+	20+
EcoRI	10	5
PstI	10	5
Buffer 3	15	5
BSA	3	1
dH2O	22	14
Total	150	50

These were incubated for 1 hour at 37º C and for 20 min heat inactivated.

Nanodrop of the minipreps from the new inoculations.

pDNA new	ng/ul	260/280
51/1	9.5	2.2
51/2	9.9	1.91
51/3	8.9	1.93
51/4	8.6	2.31
51/5	13.2	2.49
45/1	8.2	1.89
45/2	8.7	2.65
45/3	10.7	2.13
45/4	9.1	2.00
45/5	9.7	2.34
28 glycerol stock	40.4	2.23
45 glycerol stock	36.7	2.24
51 glycerol stock	45.4	2.18
52 glycerol stock	28.4	1.98

These samples of minipreps and from glycerol stocks were run on a gel in order to check if the circularised potentially recombinant plasmid is present. Again, there were no bands for any of these samples, hence we tend to believe the ligations were not successful OR/AND the transformation was faulty.

Gel extraction - of digested E and P - K812014 biobrick plasmid After gel extraction purification of the corresponding 2 kb band, the concentration showed up to around 6.6 ng/ul and 260/280 = 1.73. Given the very small concentration, a second attempt of obtaining pure pSB1C3 was performed, not successful though, only 4.9 ng/ul concentration.

- of PCR purified zeocin, 2 kb band. This was purified and the nanodrop showed a concentation of 77.6 ng/ul and 260/280 = 1.82.

Nanodrop of E, P digested cmv (from 30 of August) gave a concentration of 25.1 ng/ul, 260/280 = 1.86.

New inoculations of K812014 biobrick were prepared and left for incubation over night in the incu-shaker (37 degrees Celsius, at 200 rpm).

Wednesday 4th September

Miniprep of the K812014 biobrick inoculations showed the following concentrations:

Tube number	ng/ul	260/280
1	151	1.93
2	80.8	1.93
3	111.5	1.82
4	219.1	1.94

Samples 1 and 2 went through a preparative digest

item	ul
pDNA	50
Buffer 3	10
EcoR1	7
Pst1	7
BSA	2
Water	24
Total	100

Ligation 2

Contents	ng/ul
pSB1C3 digested & pruified	25
seo digested & purified	77
cmv digested & purified	25

CMV (25 ng/ul)	1 c (3:1 molar ratio)	2 c (2:1 molar ratio)	3 c (1:1 molar ratio)	4 c (3:1 molar ratio)
Water (ul)	1.8	3.2	8	6
Quick ligase buffer (ul)	10	10	10	10
Backbone (ul)	4	4	1	1
*Insert (ul)	4.2	2.8	1	3
Ligase (ul)	1	1	1	1
Total	21	21	21	21
Colony count	50	100+	100+	150+

Zeocin (77 ng/ul)	5 z (3:1 molar ratio)	6 z (2:1 molar ratio)	7 z (1:1 molar ratio)	8 z (3:1 molar ratio)
Water (ul)	2.1	3.3	8	8
Quick ligase buffer (ul)	10	10	10	10
Backbone (ul)	4	4	1	1
*Insert (ul)	3.9 from *	2.6 from *	1 from *	1
Ligase (ul)	1	1	1	1
Total	21	21	21	21
Colony count	30	0	1	0+

* we prepared a tube of concentration 25 ng/ul

Controls	9	10	11
Water (ul)	6	7	7
Quick ligase buffer (ul)	10	10	10
Backbone (ul)	4	4	4 of uncut
*Insert (ul)	0	0	0
Ligase (ul)	1	0	0
Total	21	21	21
Comments	Check no circular backbone	Check digestion
Colony count	0	0	0+

5 ul of each ligation was used to transform our home made competent cells. After the transformation procedure, these cell vials were entirely spread on 2 x cmp plates.

The 2 ordered oligonucleotides (1gm Oxn LF and 1gm ox LF) were annealed according to the open wet lab protocol. The thermocycler was busy at the time we booked therefore we thermocylced in the evening left in the machine at 4 degrees Celsius over night to collect the following morning.

Thursday 5th September

The pSB1C3 obtained from the postgrads was digested.

1 colony per each plate of transformation was picked and inoculated in 2 ml LB with 4 ul cmp. These were incu-shaked over night (37 degrees Celsius and 200 rpm).

PCR of zeocin and cmv was performed: 8 same reactions for zeocin, 3 same reactions for cmv and 2 controls (no pSecTag2A template). The zeocin and cmv reaction tubes contained a volume of 1.5 ul template. Primers used for zeocin: zec bb F,R and for cmv: bFW and bbRE.

Ran out of any Phusion DNA polymerase.

OxLF and OxLF Annealing oligos The annealed oligos were retrieved from the thermocycler in the morning. The entire tube contents (30 ul) underwent PCR purification - producing aprox. 30 ul pure DNA. 5 ul from this volume was run on a 3% agarose gel with both a 50 bp and 1 kb DNA ladder (120 V for 1 hour). The gel showed the annealed oligos at the right length. The left 24 ul of the pure DNA tube were prep digested while 1 ul was used for nanodroping ( 318.6 ng/ul, 260/280 = 1.84).

Prep digest of purified DNA oligos.

Component	Volume added (ul)
Sample	24
EcoRI	5
PstI	5
Buffer 3	10
BSA	2
dH2O	4
Total	50

These digests were run on a gel and the correct bands were extracted.

Friday 6th September

Did glycerol stocks and minipreps of the new transformations.

Nanodrop results of the miniprep:

	1 c	2 c	3 c	4 c	5 z	7 z
ng/ul	7	12.2	17.3	14.1	18.6	13.8
260/280	1.78	1.69	1.87	1.98	1.88	1.68

Analytical digest of the above minipreps:

Component	Volume added (ul)
pDNA	5
EcoRI	1
PstI	1
Buffer 3	1
BSA	0.5
dH2O	1.5
Total	10

These digestions were run next to uncuts but again, no bands on the gel.

Gel extraction and purification of: EcoR1 and Pst1 (E and P) digested K812014 (2 kb band corresponding to pSB1C3) was purified and the nanodrop results were: 51.1 ng/ul and 260/280 = 1.88 OxLF and OxLF annealed. Nanodrop result: 44.6 ng/ul and 0.08 for 260/280.

Saturday 7th September

Picked a total of 15 colonies from plates of potentially ligated cmv and zeocin in pSB1C3 - from both transformations (31st of August and 4th of September). Incubated overnight for 16 hours in 2 ml LB broth and 4 ul chloramphenicol (cmp). Made new glycerols stocks using 0.5 ml of the inoculated volume. Minipreped the 15 inoculations but the nanodrop of these showed concentrations of below 20 ng/ul.