Gibson assembly round 3 #
Today I am preparing Gibson assembly reactions for the VR inserts listed below.
| Fragment |
|---|
| VR-6 |
| VR-7 |
| VR-8 |
| VR-9 |
| VR-11 |
| VR-12 |
| VR-13 |
| VR-15 |
| VR-16 |
| VR-17 |
| VR-18 |
| VR-19 |
| VR-20 |
| VR-21 |
| VR-22 |
| VR-23 |
| VR-25 |
| VR-26 |
| VR-27 |
| VR-28 |
| VR-30 |
| VR-31 |
Made new 1.33x Gibson master mix according to the recipe in the
Gibson assembly spreadsheet and
created reactions according to the 9-6-21 spreadsheet. After incubating reactions for 1 hour at 50C I transformed NEB
chemically competent cells according to the provided protocol and
then plated cells by streaking with platinum loop until that unexpectedly broke during heating and after with 100ul of transformed culture and spreading with beads.
LiCl purification of completed midi preps #
Yesterday when nanodropping the completed mini-preps the results indicated strong RNA contamination. So today I am completing a LiCl purification for all samples. I am following the lab protocol which is listed as part of the midi prep protocol and is included below.
Resuspend pellets in 1 ml TE, add RNase A to a final concentration of 20 μg/ml.
Incubate at 37°C for 30 min.
10. Add 1 ml of 10M LiCl, mix by inverting 4-6 times.
11. Incubate for >= 20min. at -20°C.
12. Centrifuge at 14,000rpm for 10 min at 4°C.
13. Transfer the supernatant to a clean tube.
14. Add 2.2 volume of ethanol 100%; mix by inverting.
15. Centrifuge at 14,000 rpm for 10 min at 4°C.
16. Wash twice with 70% ethanol.
17. Dry the pellet and dissolve in 500µl of 10 mM Tris HCl pH 7.5.
At step 14 I accidentally added 70% ethanol instead of 100%. This lead to extreme difficulty in precipitating the DNA. Megan also mentioned that the EtOH in the brown bottle is 95% not 100% EtOH. In the morning tomorrow will do one last round of precipitation and pull out what I can from each sample.