Team:UCLA/Notebook/Spider Silk Genetics/8 July 2015

iGEM UCLA




7/8/2015

pSB1C3 Digestion

  • Digested 600 ng of pSB1C3 linearized backbone in 50 uL reaction with EcoRI and PstI.
  • Used the kit provided linear pSB1C3 (25 ng/uL)
Volume (uL)
EcoRI 1
PstI 1
10x NEBuffer 2.1 5
DNA (600 ng) 24
ddH2O 19
Total 50
37 C 1.5 hrs
65 C 20 min
12 C hold

Gel Purification of MaSp Seq AB and ICA 3-mer

  • Ran samples from 7/7/2015 on 1.5% TAE gel; used 2 uL 50 bp ladder.
Fig. 1 Gel image of ICA 3-mer (expected 406 bp), and MaSp2 Seq AB (expected 170 bp)
  • Excised bands corresponding to the 3-mer, and 64 C MaSp2 Seq AB. There was no product present at the 61 C annealing for MaSp2 Seq AB.
  • Purified using Qiagen Kit.
    • MaSp2 Seq AB: 64.75 ng/uL, A: 2.33
    • ICA 3-mer: 38.21 ng/uL, A:2.29

Digestion of 3-mer, 6-mer, MaSp2 Seq AB

  • Digested 10 uL of each DNA species, regardless of concentration.
  • Digested in 50 uL reaction, using 1 uL each of EcoRI, PstI, using 10x NEBuffer 2.1.
  • Digestion conditions same as above section.

PCR purification

  • PCR purified using Zymo Clean and Concentrator -5, for pSB1C3 backbone, MaSp2 Seq AB, 3-mer, and 6-mer.
    • All of these were digested with EcoRI and PstI.
Concentration (ng/uL)
3-mer 14.28
6-mer 6.81
MaSp2 Seq AB 24.31
pSB1C3 53.89

Electro-transformation of BBa_K525998

  • BBa_K525998 is a T7 promoter expression vector that is supplied with the registry kit. It is located in well 3M.
  • Reconstituted the DNA in 15 uL ddH2O.
  • Electro-transformed into cells.
    • Arc-time: 5.6 ms
  • Plated overnight.

Preparation of BL21(DE3)

  • Streaked overnight culture of BL21(DE3) cell onto LB plate for single colonies.
  • Plan to make chemically competent cells.