Team:Caltech/Week 6

July 18

Colonies 1-6 from Gibson plates; 7 and 8 are reswabs of two colonies. From left to right: DNA ladder,(PNT001; colony 1; vector), P2v, P3v, P4v, P5v, P6v, P7v, P8v, (PNT001; colony 1; insert), P3i, P4i, P5i, P6i

from left to right: DNA ladder, (PNT001; reswab 7; insert), P8i, (16s; colony A; vector), 16Bv, 16Cv, (16s; A; insert), 16Bi, 16Ci

Colony PCR of 16s plasmids, for vector and insert
Gel of short PCR parts to confirm they are being amplified
Pulsed field gel with a larger amount of soil sample DNA
Retransform pSB3K5 into Xl-10s

Results

Gels show mostly expected single bands

July 19

PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control

Results

pSB3K5 plate had 0 colonies.
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.

lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10

Gibson PCR concentrations

July 20

Pulse field gel of soil-extracted DNA, in preparation for fosmid insertion
PCR parts for Gibson assembly of pNT001, pNT003 and positive control
Redo Gibson assembly of pNT001 using a smaller total mass of DNA. Gibson assemble GFP from Joe that is known to have worked before as a positive control
Try transforming pSB3K5 with less DNA and as a liquid culture
Prepare minimal media plates with chemical layer

Results

PCR concentrations

Minimal media plates: BPA and DDT plates came out wrong. Will plate DDT cultures anyway and redo.

Minimal media plates. Left to right: 17a-ethinylestradiol, DDT, BPA

July 21

Gel extraction of 40kb LA River sample DNA from pulse field gel
Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working
Run Gibson assembly of Joe's GFP and pNT001 and stop at different time points. Run on a gel to see what size DNA there is at different times in the reaction
Try "step-wise" Gibson. Add the insert for pNT001 (3 ul) into a 10 ul reaction and incubate at 50˚C for 10 or 20 minutes before adding to a premade Gibson mix of just the vector (2 ul). Continue incubating until a total of 1 hour has passed. This may help with the self-ligation of the vector by giving the insert time to assemble if it works
Plate DDT and estradiol cultures on minimal media plates

Results

lane 1 NEB 2-log ladder, 2 GFP 0 min, 3 GFP 5 min, 4 GFP 10 min, 5 GFP 15 min, 6 GFP 30 min, 7 GFP 60 min, 8 blank, 9 pNT001 0 min, 10 pNT001 5 min, 11 pNT001 10 min, 12 pNT001 15 min, 13 pNT001 30 min, 14 pNT001 60 min, 15 NEB 2-log ladder. Triangles represent increasing time (not to scale)

Gibson Plates

July 22

Gave a presentation of our project so far to Dr. Murray's bio subgroup.
Sent off pNT001 and GFP Gibson colonies for sequencing.
Attempted to PCR pNT001 insert from PCR'd products (part plus overlapping primers)

Results

Gibson Plates

Transform Plates

lane 1 NEB 2-log ladder, 2-6 pNT001insert

Miniprep concentrations

PCR of the pNT001 insert was successful. The bright band around 700 bp is the correct length for the insert.

July 23

Results

Sequencing results are back. pNT001 sample 1 was the only one with a good alignment. Blasted both GFP colonies and they contained vector only.
PFGE-extracted DNA concentrated by evaporation centrifuging; nanodrop results: 9-1: 11.4ng/uL; 10-2: 29.7ng/uL