Team:UCLA/Notebook/Spider Silk Genetics/20 July 2015
Contents
7/20/2015
BsaI Digestion of MaSp2 AB, BC, CA
- Same protocol as 7/13/2015.
Sub-cloning M2-9(1C3) into BBa_K525998: Preparation
- Digest 10 uL of plasmid (~2.8 ug) using 1 uL XbaI and 1 uL of PstI in a 50 uL reaction.
- Used NEBuffer 2.1
- Digest 37 C for 1.5 hrs, heat kill 65 C for 20 min.
- Gel-purify resulting product.
Cloning M2-12 into pSB1C3: Preparation
- Digest 15 uL PCR product using 1 uL each of EcoRI and PstI in a 50 uL reaction
- Used NEBuffer 2.1
- Digest 37 C for 1.5 hrs, heat kill 65 C for 20 min.
- Gel-purify resulting product.
Gel Purification Results
- Cast 1.5% TAE gel. Used 2 uL each of 100 bp ladder and 1 kb ladder from NEB.
- Excised all the above indicated bands for gel purification.
Ligation
- M2-9(1C3) into BBa_K525998, and M2-12 into pSB1C3.
- Used [insilico.uni-duesseldorf.de ligation calculator] to determine amounts of DNA to add.
- vector size: 2200 bp.
- vector amount: 50 ng.
- insert size:
- M2-9: 1018 bp
- M2-12: 11224 bp
- vector to insert ration: 1 to 3
- Calculated for 50 ng of vector, add:
- 69.41 ng for M2-9
- 83.45 ng for M2-12.
- Ligation in 20 uL volumes.
- Ligate 25 C for 25 min, heat kill 65 C for 10 min.
Transformation
- Transformed M2-9(T7) into chemically competent BL21(DE3) cells.
- Transformed M2-12(1C3) into chemically competent BL21(DE3) cells.
- Previous tranformations of M2-mers(1C3) were performed using DH5(alpha) cells.
- Rescues were at 37 C for 30 min.
Glycerol Stock Reconstitution
- Plated M2-AB, M2-BC, M2-CA, and M2-SeqAB from glycerol stocks.
- Aim to grow culture for DNA prep.