Team:UCL/Labbook/Week13

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Lab Weeks

Week 13

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)

Mammalian Lab

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