"
Team:BYU Provo/Notebook/CRISPR/febapr
From 2014.igem.org
BYU 2014 Notebook |
||||||||||||
| ||||||||||||
4/07/2014 - Garrett Jensen.
- This week we worked Monday on our circuit design paper. The assignment is saved on my computer in the igem folder.
4/09/2014 - Garrett Jensen.
- today we worked on the circuit assignment more. We did not make primers for inserting our own spacers because dr grose told us not to just yet.
- Started doing plasmid preps of the promoters in the igem registry. We removed some of the primers from the igem plates and transformed them into E. coli. After this we plated them on lb/amp and incubated overnight. Friday we will harvest our plasmids from these cells in preparation for moving them into n multiformis.
4/11/2014 - Garrett Jensen
-today we are harvesting the plasmids from E. coli usig the plasmid DNA purification kit. We are following the instructions desi gives us to the letter!
4/27/14 - Garrett Jensen.
Today we worked on amplifying our plasmid from the igem registry so that we have it ready to use when we get our crispr ready to move into e coli.
- Using E coli DH5α we added in 1 µL of igem plasmid, incubated on ice for 2 min
- Heat shock at 42C for 60 sec.
- Recovered on Ice for 5 minutes
- Incubated for 30 minutes at 37C
- Plated 100 µL on a plate with chloramphenicol. 400 µL on another plate with chloramphenicol. The plasmid is very concentrated, so transformation is usually very efficient. Plating all 500 µL of our bacteria would give us a whole lawn of bacteria, so we only will plate 100 so we can get individual colonies. We will need to pick a colony and start an overnight from that to do plasmid preps from next class
4/28/14- Garrett Jensen.
-I went in today to start our overnight, but no bacteria grew. Desi said we might be able to plate our cells after 30 minutes incubation but she always does 90. 30 must not have worked because none of our cells grew, but Jordan's did and he incubated his for longer.
- ○ ***NOTE: when transforming e coli with a plasmid that has resistance to anything but ampicillin you need to incubate it for 60-90 minutes before plating.
