Friday 26^th August

Lab work

Visualization

Purification of pPS16-003 and pPS16_004 ligation products

By Mathilde

Ligation products pPS16-003 and pPS16_004 frome the day before were purificated according to the usual protocol

Q5 high fidelity PCR of pPS16_003 - pPS16_004 ligation product

By Mathilde

The Q5 PCR was conducted following the exact same protocol than the 25/08/2016 on the ligation product from the day before.

Migration Results

The two bands are at the expected size (1860 pb), so the fragment 2 (16-003 and pPS16_004 ligation product) is purified.

Purification of fragments 1(16-001 + pPS16_002) and 2 (16-003 + pPS16_004)

By Mathilde

The purification was made following the usual protocol. Each of those purification products were quantified on nanodrop.

Enough DNA quantity was obtained to perform a Gibson assembly on segments 1 and 2.

Gibson Assembly of segments 1 and 2

By mathilde

Two preparations were made :

• the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix
• the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water

Gibson products were incubated 1h at 52°c.

Transformation in DH5α with the 1-2 Gibson assembly product

By Mathilde

The transformation was performed following the usual protocol with 2µL of plasmid.

• 50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL)
• 350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL)

Petri cultures were incubated at 37°c overnight.