Team:UCL/Labbook/Week14

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Lab Weeks

Week 14

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
water (ul) 1.8 3.2 8 6
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
water (ul) 1.8 3.2 8 6
Colony count 30 0 1 0+