Contributors: Chloe
Q5 PCR reclone, less DNA (p. left of this spread, no date written)
Left to run overnight.
| Component | xmm10 (✱) | ymm10 |
|---|---|---|
| Q5 reaction buffer | 50 µl | 50 µl |
| GC enhancer | 50 µl | 50 µl |
| dNTPs | 5 µl | 5 µl |
| Template DNA (1 in 1000 dilution) | 10 µl | 40 µl |
| Q5 polymerase | 2.5 µl | 2.5 µl |
| Water | 357.5 µl | 327.5 µl |
1 in 1000 dilution made from 999 µl water + 1 µl MSA [?]. 47.5 µl xmm10 + 1.25 µl primer, ×5 replicates, plus a negative control; 47.5 µl ymm10 + 1.25 µl primer, ×5 replicates, plus a negative control. Primers: End For1, Start Rev1. Negative control: no primers, with 2.5 µl extra water added.
Gel extraction setup
Ladder made: 20 µl ladder + 80 µl loading dye + 100 µl water. Gel: 250 ml 1× TAE buffer + 2 g agarose + 12.5 µl gel red.
Three stored gel products used — the longest bands from the 20 August gel — labelled 1, 2, 3. Elution run for 6 minutes rather than the usual 5. 10 µl loading dye added to each sample before loading.
Gel extraction results, Nanodrop, and miniprep 6
(No date written on this page — placed here as a direct continuation of the gel extraction above.)
Lanes: ladder (25 µl) | −ve ✱ | A ✱ | B ✱ | C ✱ | D ✱ | E ✱ | −ve | A | B | C | D | E | sample ② | ladder (25 µl); 50 µl loaded. A note by sample ② reads “sample didn’t get in well.” Only one product was visible — cut out and put in the fridge, possibly because it wasn’t mixed well.
Gel extraction Nanodrop (three unlabelled numbers per sample, presumably conc./260-280/260-230):
| Sample | Values |
|---|---|
| ① | 7.529, 2.179, 1.108 |
| ② | 5.774, 2.884, 0.493 |
| ③ | 5.671, 4.197, 0.531 |
Miniprep 6, TOP10 CxnA + CxnA-His, 5 ml of overnight culture used. One Nanodrop line is scribbled out.
| Sample | Conc. | 260/280 | 260/230 |
|---|---|---|---|
| CxnA | 35.731 | 1.793 | 2.283 |
| CxnA-His | 436.849 [?] | 1.908 | 2.192 |
CxnA-His’s concentration and 260/230 figures are overwritten in the original — transcribed as written, flagged rather than guessed at.
Heather repeats the PCR
Repeated with Q5 ymm10 (10 min [?]), primers End For1 + Start Rev1 → successful replicates, plus one unused replicate with a smear. A note underneath asks whether the primers themselves might be wrong.