Team:Caltech/Week 7

July 24

Started overnight cultures of pSB3K3

July 25

PCR'd parts for pNT002: R0040, K123001, B0014, pSB4A5
PCR'd GFP control insert and pNT003 insert
Gibson assembly and transformation of pNT003 with a negative control
Nanodrop of evaporation concentrated PFGE DNA: 9-1: 11.4ng/uL; 10-2: 29.7ng/uL
Attempt at packaging above DNA with fosmid kit extract, but enzyme wasn't heat inactivated as par procedure
50 mL of phage dillution buffer made for fosmid kit
Evaporation concentrated more PFGE DNA. Nandrop results: 9mix: 14.3ng/uL; 10mix: 2.2ng/uL
Overnight ligation of 9mix and (10mix + 10-2) to redo fosmid packaging, at 16°C

Results

Decided not to use the pNT003 PCR'd insert, as the band of the correct length was much fainter than a lower length band. Chose not to do Gibson assembly of the positive GFP control, despite not having any green colonies last week, instead focusing on assembling pNT002 and pNT003.

lane 1 NEB 2-log ladder, 2-4 pSB4A5, 5-7 R0040, 8-10 K123001, 11-13 B0014

lane 1 NEB 2-log ladder, 2-4 pNT003 insert, 5 R0010, 6 K123003, 7 B0014, 8 blank, 9-11 GFP insert, 12 GFP constitutive promoter, 13 GFP, 14 GFP terminator, 15 NEB 2-log ladder

PCR concentrations

PSB3K3 miniprep concentrations

July 26

Packaging of 9mix and (10mix + 10-2) ligations
Attempted various methods of spreading chemical solution on minimal media plates
16s PCR
Ran 20 ul Gibson reaction of pNT003 and a negative control. PCR purified these using the Qiaquick kit. Ran an Spe1 digest on part of the reaction. If the plasmid had formed, a cut with Spe1 would create a band of around 5kb.
Plated enrichment cultures on LB

Results

The gel gave no bands in any lane, with or without the restriction digest. pNT003 + without the Spe1 digest had a faint smear, the other lanes had nothing. Chose to transform anyway

All enrichment cultures but one showed growth on LB.

July 27

Grew up cells for titering of packaged fosmids; reached OD600 of 0.974
Redid 16s PCR
Attempted to PCR the inserts for pNT002 and pNT003 together from their component parts. Did a gel extraction to get the correct length product
PCR pSB3K3 to linearizer it for Gibson assembly
Continued attempts to spread chemical solution on minimal media plates
Minimal media transfers

Results

lane 1 NEB 2-log ladder, 2-3 negative controls, 4-6 LA River sample 9, 7-9 LA River sample 10, 11-12 pET53DEST

lane 1 NEB 2-log ladder, 2-4 pSB3K3

The 16s PCR from yesterday, shown in the gel today, had a band in the first lane of negative controls, indicating contamination.
The pSB3K3 amplification failed. The bands indicated a much lower length than expected. After doing an alignment in Geneious, it appears our pSB3N5 primers we ordered for pSB3C5, which we are no longer using because of our competent cell strain, do not work with earlier versions of the 3 origin plasmids.
Both the negative control and experimental of PCR purified Gisbson assembly from yesterday of pNT003 had 0 colonies. The transformation with 1 ul pUC 19 had no colonies.

BPA solutions fail to form uniform layers when poured on top of agar, but form a uniform layer when poured directly onto a clean plate.

July 28

Plate packaged fosmids to obtain titer
Set up enzyme binding assay of p450s
Try out plating chemicals on bottom of plates with agar bacteria suspension on top
Try conventional assembly of pNT002 and pNT003
Find positive control for transformation, test transformation
Run gel of 16s, DpnI digest, purify, gibson