Contributors: Chloe
Q5 backbone PCR
Conditions carried over from 14 July, varying template DNA amount: 1 µl, 0.5 µl, and 0.25 µl.
Master mix (mm10): 50 µl Q5 reaction buffer, 50 µl GC enhancer, 5 µl dNTPs, 2.5 µl Q5 polymerase. Split into three groups (×3 replicate tubes each), 10.75 µl mix per tube, 1.25 µl primers per tube:
| Template DNA | Replicates | Mix (µl) | Primers (µl) | Water (µl) [?] |
|---|---|---|---|---|
| 1 µl | ×3 | 10.75 | 1.25 | 35.75 |
| 0.5 µl | ×3 | 10.75 | 1.25 | 36.25 |
| 0.25 µl | ×3 | 10.75 | 1.25 | 36.5 |
Primers: End For1 + Start Rev1, plus a negative control (no primers).
Loading the PCR gel
10 µl loading dye added to the PCR product; 50 µl loaded. Gel: 0.8% agarose → 250 ml TAE buffer, 12.5 µl gel red, 2 g agarose.
Lanes: 25 µl ladder [?] | −ve | 1 µl A | 1 µl B | 1 µl C | 0.5 µl A | 0.5 µl B | 0.5 µl C | 0.25 µl A | 0.25 µl B | 0.25 µl C | ladder.
A note on the gel photo reads “these three products stored/cut out” (arrow). Open questions noted on the page itself: too much template DNA? Or a bad batch of ladder?