Congo red screen, miniprep, and digestion of colonies 3 & 5

Contributors: Esther, Tank

Congo red screen (CMC plates)

0.5 mg/ml Congo red poured over the plate until the surface was covered, left 15 minutes, then poured off into the Congo-red waste Duran. Washed with 1 M NaCl for 15 minutes and poured off.

Zones of inhibition:

The label on the last row is hard to read in the original — transcribed as “CxnA-H87” with a [?] in the source; whether this is CxnA-His or something else isn’t confirmed here.

Miniprep — colonies 3 & 5

Overnight cultures of colonies 3 and 5. Each 10 ml culture split into two; one pelleted for immediate use, the other left in the freezer as a backup for a repeat miniprep. Sample 5’s Buffer P2 step precipitated but was used anyway.

Restriction digestion — colonies 3 & 5 vs CxnA

2 µl CutSmart + 2 µl DNA + 0.5 µl EcoRI + 15.5 µl water (20 µl total) for each of: colony 5 (cut + uncut, 16 µl water, no EcoRI for the uncut), colony 3 (cut + uncut), and CxnA original (cut + uncut). 5 µl loading dye added to each; 25 µl of each loaded on a small gel.

The notebook literally reads “put in the microwave for 55 seconds” at this step — transcribed as written, not corrected.

Digest gel results & sequencing prep

(Same session — the source pages carry no separate date but are grouped with 04/09/26 by page position.)

Gel layout: ladder (10 µl) | 5 cut | 5 uncut | 3 cut | 3 uncut | CxnA cut | CxnA uncut (25 µl each).

• Colony 5: not high enough concentration for two bands to resolve.
• Colony 3, cut: two bands, but running larger than expected.

Sequencing prep (target: 25 ng/µl final, 40 µl final volume):

• Colony 3 (135 ng/µl): V = 25 × 40 / 135 = 7.4 µl DNA + 32.6 µl water
• Colony 5 (38 ng/µl): V = 25 × 40 / 38 = 26.3 µl DNA + 13.7 µl water
• 10 µl of cPCR-R + cPCR-F at 3.2 µM [?]

Heather’s manganese-gradient epPCR (same session)

Error-prone PCR run across a manganese concentration series — 0, 0.01, 0.02, 0.05, 0.1 — to find the mutation-rate sweet spot.

• Sizing gel: 10 µl run for sizing → ~2.5 kb, the desired size.
• Big gel lanes: ladder | 0 | 0.1 | 0.02 | 0.05 | 0.01 (as written in the notebook). The 0.02, 0.05, and 0.01 lanes were bracketed and cut out of the gel, then stored in the freezer.
• Small gel lanes: ladder | 0 | 0.01 | 0.02 | 0.05 | 0.1.

This is almost certainly where the 0.01/0.02/0.05 mM manganese conditions carried into the 7 September gel extraction came from — the same three concentrations reappear there as the ones being gel-extracted.