Team:University College London/LabBook/Week12

11.4

Monday 27.8.12

Aim: To find the right concentration of W3110 E. coli cells in LB that would be later used to streak out on the agar plates to obtain an optimal amount of colonies.

Methods: 1. The colony was inoculated the night before in 10ml of LB plus 10 ul CMP 2. The day after 5 dilutions were made from the sample 3. First solution - 1ml of the original solution & 4ml of the LB Second – 1ml of the first solution & 4ml of the LB Third – 1 ml of the second solution & 4ml of the LB Fourth – 1 ml of the third solution & 4ml of the LB Fifth – 1ml of the fourth solution & 4ml of the LB

Tuesday 28/08

Aim: (on Monday grew WNu o/n in prep for miniprep. This miniprep will be used as a template for pcr of a 2nd nuclease.

Not the synthesised staph aureus nuclease (Plan A). The Wnu Nuclease is considered our plan B.

Methods: (10ul cells, 10ul, amp, 10ml (LB). Wnu cells were obtained from a glycerol stock.

Optimal number of colonies was achieved using the fourth solution/dilution (24 colonies). Using the dilution number 4 we plated out 12 plates.

12.2

when we used diluted dinoccocus 1 10 times, second 100

PCR did not work. PCR was preformed using NEB high fidelity polymerase. The following reaction was set up in 50uL:

All primers were prepared to a concentration of 1pmol.uL, from 100pmol.uL stocks. Primers ordered from MWG operon.

Forward primer:

STF1: 5'-atggggccaaaagctaaagctgaagcc-3'

Reverse primer:

ST2R: 5'-tcactgtgcagcgtcctgcg-3'

With Biobrick prefix/Suffix:

Forward primer:

STF3 5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3'

Reverse primer:

ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'

Component 20 µl Reaction 50 µl Reaction Final Concentration Nuclease-free water to 20 µl to 50 µl 5X Phusion HF Buffer 4 µl 10 µl 1X 10 mM dNTPs 0.4 µl 1 µl 200 µM 10 µM Forward Primer 1 µl 2.5 µl 0.5 µM 10 µM Reverse Primer 1 µl 2.5 µl 0.5 µM Template DNA variable variable < 250 ng DMSO (optional) (0.6 µl) (1.5 µl) 3% Phusion DNA Polymerase 0.2 µl 0.5 µl 1.0 units/50 µl PCR

1. EXP 11.4 (Repeat inocculation of nuclease + curli ligation transformations) Monday 27/08

Aim: To inoculate colonies from ligation transformation plates, prepared on the 23rd and 24th of August.

Inoculation was repeated due to no growth the first time round.

1. EXP 12.2 (PCR for irrE) Monday 27/08

Colony PCR was preformed on Deinococcus radiodurans. 5uL of colony matter was resuspended in 10uL dH2O and added to boiling water in a screw top vial,and left to return to ambient room temperature.

1uL was used as PCR template.

PCR was preformed using NEB high fidelity polymerase. The following reaction was set up in 50uL:

All primers were prepared to a concentration of 1pmol.uL, from 100pmol.uL stocks. Primers ordered from MWG operon.

Forward primer:

STF1: 5'-atggggccaaaagctaaagctgaagcc-3'

Reverse primer:

ST2R: 5'-tcactgtgcagcgtcctgcg-3'

With Biobrick prefix/Suffix:

Forward primer:

STF3 5'-gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc-3'

Reverse primer:

ST4R 5'-gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg-3'

Component 20 µl Reaction 50 µl Reaction Final Concentration Nuclease-free water to 20 µl to 50 µl 5X Phusion HF Buffer 4 µl 10 µl 1X 10 mM dNTPs 0.4 µl 1 µl 200 µM 10 µM Forward Primer 1 µl 2.5 µl 0.5 µM 10 µM Reverse Primer 1 µl 2.5 µl 0.5 µM Template DNA variable variable < 250 ng DMSO (optional) (0.6 µl) (1.5 µl) 3% Phusion DNA Polymerase 0.2 µl 0.5 µl 1.0 units/50 µl PCR

PCR successful, band corresponding to 930bp length.

PCR cleanup of Irre followed with Anachem PCR cleanup kit.

Concentration determined by nanodrop: 9ng.uL-1

12.3