VR 17, 20 and 27 mini preps #
Came into lab at 6:10 am to start minipreps of liquid cultures I started yesterday. Followed standard miniprep protocol. Finished protocol at 10:18 am and ODed all samples, results of which are shown in the table below.
| Sample | Colony | ng / ul (40 ul) | ng / ul (final volume) |
|---|---|---|---|
| VR-17 | 1 | 1218 | 609 |
| VR-17 | 2 | 1535 | 767.5 |
| VR-17 | 3 | 1157 | 578.5 |
| VR-17 | 4 | 1237 | 618.5 |
| VR-17 | 5 | 1505 | 752.5 |
| VR-17 | 6 | 1723 | 861.5 |
| VR-17 | 7 | 1203 | 601.5 |
| VR-17 | 8 | 1364 | 682 |
| VR-20 | 1 | 1104 | 552 |
| VR-27 | 1 | 1323 | 661.5 |
| VR-27 | 2 | 1527 | 763.5 |
| VR-27 | 3 | 1405 | 702.5 |
| VR-27 | 4 | 1209 | 604.5 |
KpnI digest and gel #
Set up the KnpI digests described in the table below for each completed miniprep sample. Samples that have the insert should show two bands when run on a gel.
| Sample | Colony | DNA volume (ul) | 10x Cutsmart buffer | KpnI | H20 |
|---|---|---|---|---|---|
| VR-17 | 1 | 1.642036125 | 2 | 1 | 15.35796388 |
| VR-17 | 2 | 1.302931596 | 2 | 1 | 15.6970684 |
| VR-17 | 3 | 1.72860847 | 2 | 1 | 15.27139153 |
| VR-17 | 4 | 1.616814875 | 2 | 1 | 15.38318513 |
| VR-17 | 5 | 1.328903654 | 2 | 1 | 15.67109635 |
| VR-17 | 6 | 1.160766106 | 2 | 1 | 15.83923389 |
| VR-17 | 7 | 1.662510391 | 2 | 1 | 15.33748961 |
| VR-17 | 8 | 1.46627566 | 2 | 1 | 15.53372434 |
| VR-20 | 1 | 1.811594203 | 2 | 1 | 15.1884058 |
| VR-27 | 1 | 1.511715797 | 2 | 1 | 15.4882842 |
| VR-27 | 2 | 1.309757695 | 2 | 1 | 15.69024231 |
| VR-27 | 3 | 1.423487544 | 2 | 1 | 15.57651246 |
| VR-27 | 4 | 1.654259719 | 2 | 1 | 15.34574028 |
After incubating all samples for 45 mins at 37C ran products plus a control lane for each sample on 1% 1x TAE agarose gel at 120V for 45 minutes. Lane layout described in the table below.
| Lane | Sample | Lane | Sample | Lane | Sample | Lane | Sample |
|---|---|---|---|---|---|---|---|
| 1 | 1kb Ladder | 9 | 17-4 + KpnI | 17 | 17-8 + KpnI | 25 | 27-2 + KpnI |
| 2 | 17-1 | 10 | 17-5 | 18 | 20-1 | 26 | 27-3 |
| 3 | 17-1 + KnpI | 11 | 17-5 + KpnI | 19 | 20-2 + KpnI | 27 | 27-3 + KpnI |
| 4 | 17-2 | 12 | 17-6 | 20 | NA | 28 | 27-4 |
| 5 | 17-2 + KpnI | 13 | 17-6 + KpnI | 21 | 1kb ladder | 29 | 27-4 + KpnI |
| 6 | 17-3 | 14 | 17-7 | 22 | 27-1 | ||
| 7 | 17-3 + KpnI | 15 | 17-7 + KpnI | 23 | 27-1 + KpnI | ||
| 8 | 17-4 | 16 | 17-8 | 24 | 27-2 |
Most samples digested but also looks like VR-20 sample may not actually have the insert. Thinking will still send for sequencing to make sure / see what it is.