Team:UCL/Labbook/Week15

Colony counts of the re-transformation of ligations:

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

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):

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

Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).

Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).

Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands: - For zeocin 2 bands: one of 1037 bp and another of 783 bp - For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp

The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul). These cuts were run on a gel, next to uncuts.