VR insert IVT #
Ran IVT with completed inserts that had enough DNA to spare to use in an IVT. Reaction descriptions are listed in the table below.
| Plasmid | DNA (ul) | 5x Transcription buffer (ul) | DTT | rNTP (ul) | npH20 (ul) | Pol Species |
|---|---|---|---|---|---|---|
| pFC9 | 2 | 6 | 3 | 0.6 | 18.4 | T7 |
| pFC9VR13 | 0.8163265306 | 6 | 3 | 0.6 | 19.58367347 | T7 |
| pFC9VR1 | 2.739726027 | 6 | 3 | 0.6 | 17.66027397 | T7 |
| pFC9VR10A | 3.75 | 6 | 3 | 0.6 | 16.65 | T7 |
| pFC9VR11A | 1.612903226 | 6 | 3 | 0.6 | 18.78709677 | T7 |
| pFC9VR14 | 1.840490798 | 6 | 3 | 0.6 | 18.5595092 | T7 |
| pFC9VR2 | 2.298850575 | 6 | 3 | 0.6 | 18.10114943 | T7 |
| pFC9VR4A | 3.03030303 | 6 | 3 | 0.6 | 17.36969697 | T7 |
| pFC9VR5A | 2.197802198 | 6 | 3 | 0.6 | 18.2021978 | T7 |
Then ran products on TBE gel without EtBR for 2 hours at 60V. Then post-stained the gel for 45mins in TBE with EtBr.
VR vector BglII digestion #
Digested 200 ng fragments from vectors with 0.2 ul BglII for 1hr in the hot room at 37C.
There is a subtle shift in band heights that may be due to under digestion but Fred suggested this may also be due to sequences differences resulting in different DNA secondary structures subtle band height differences.