Team:UCL/Labbook/Week15

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Lab Weeks

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Week 15

Bacterial Labs

Monday 9th September

Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.

Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.

Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.

LIGATION 3:

pSB1C3 digested and purified, concentration = 25 ng/ul

zeo digested and purified conc = 77 ng/ul

CMV digested and purified conc = 25 ng/ul

Zeocin (77 ng/ul) 3 to 1 (mass ratio) [z1] 6 to 1 (mass ratio) [z2]
water (ul) 6 4
Quick ligase buffer (ul) 10 10
backbone (ul) 2 2
insert (ul) 2 4
ligase (ul) 1 1
Total 21 21

Cmv (25 ng/ul) 3 to 1 (mass ratio) [c3] 6 to 1 (mass ratio) [c4]
water (ul) 2 3
Quick ligase buffer (ul) 10 10
backbone (ul) 2 1
insert (ul) 6 6
ligase (ul) 1 1
Total 21 21

Controls 5 ctrl check no circular backbone 6 ctrl check digestion process 7 ctrl (uncut backbone)
water (ul) 7 18 18
Quick ligase buffer (ul) 10 0 0
backbone (ul) 3 3 3 of uncut backbone
insert (ul) 0 0 0
ligase (ul) 1 0 0
Total 21 21 21

These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.

Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).

These were incubated overnight and then minipreped.

Tuesday 10th September

Colony counts of the re-transformation of ligations:

Plate Colony counts
2z from 31/08 100
5z from 4/09 150+
6z from 4/09 80+
7z from 4/09 100
8z from 4/09 0
9z - ctrl from 4/09 40
11z - ctrl from 4/09 0

MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock.

Colony count - 90% - 100+ and 10% 80 colonies.

Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.

The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.

The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes. The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.

Building up the competent cell stock using the home-made competent cells

Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread

Plate number Drug Volume competent cells (ul) Colony count
I 1000 X CMP 20 60
II 500 X CMP 20 15
III 250 X CMP 20 0
IV N/A 20 100+
V 1000 X CMP 20 10
VI 500 X CMP 20 3
VII 250 X CMP 20 0
VIII N/A 20 100+
IX 1000 X CMP 100 from T vial 15
X N/A 100 from T vial 100+
I 1000 X CMP 20 60

Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.

New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.

Transformation number 3:

We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.

The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.

After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.

On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.

Wednesday 11th Sepetmber

Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.

Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):

Falcon Tube no. Content: Plate & Colony ng/ul 260/280
1 2z.1 54.0 1.82
2 2z.2 28.3 2.21
3 2z.3 25.6 3.03
4 2z.4 20.3 3.16
5 2z.5 13.4 3.08
6 2z.6 15.7 2.61
7 2z.7 8.9 14.17
8 2z.8 14.8 4.08
9 5z.1 64.9 1.70
10 5z.2 36.5 1.93
11 5z.3 16.6 2.01
12 5z.4 32.1 2.22
13 5z.5 23.6 2.50
14 5z.6 53.3 1.87
15 5z.7 33.7 2.22
16 5z.8 50.1 2.06
17 6z.1 74.8 1.71
18 6z.2 32.6 1.82
19 6z.3 26.0 2.25
20 6z.4 42.5 2.15
21 6z.5 30.8 2.02
22 6z.6 27.3 2.23
23 6z.7 38.0 2.15
24 6z.8 43.5 2.10
25 7z.1 62.7 1.80
26 7z.2 31.7 1.89
27 7z.3 26.4 1.88
28 7z.4 21.4 2.12
29 7z.5 13.1 2.28
30 7z.6 27.2 2.22
31 7z.7 - -
32 7z.8 33.0 2.28

Colony counts of the transformed cells with the ligation prepared on the 9/09

Plate Colony Count
Z1 12
Z2 0
C4 1 (+small colonies)
5 50
6 50
7 17
pSecTag2A 10

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