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 miniprepping.

Preparative digest of zeocin and K812014 plasmid

These were incubated for 1 hour at 37ºC and for 20 minute heat inactivated.

Nanodrop of the minipreps from the new inoculations.

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°C, at 200 rpm).

Wednesday 4th September

Miniprep of the K812014 biobrick inoculations showed the following concentrations:

Samples 1 and 2 went through a preparative digest

Ligation 2

* we prepared a tube of concentration 25 ng/ul

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.

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:

Analytical digest of the above minipreps:

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