Team:UCL/Labbook/Week15

Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.

After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.

Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.

Ligation 4

* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;

** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex. These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.

Transformation 4

Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific transformation protocol for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant. 3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.

Friday 13th September

PCR of bb zeocin using Taq PCR kit New England Biolabs in 50 ul reaction volume.

Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09

Accomplished tasks asked by Darren:

- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.

- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.

- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).

- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.

Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band).

Saturday 14th September

Colony counts of the transformation prepared on 12th and 13th of September.

8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.

Nanodrop result of these minipreps – data from the next day

The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.

PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.

A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.

Sunday 15th September

Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.

From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.

Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):