User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/Entry Base: Difference between revisions

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Revision as of 11:50, 24 May 2011

AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

Objective

Subclone BSA into pTXB1

  • Use NheI and SapI cloning sites. This will fuse the BSA to a C-terminal intein and chitin binding domain (CBD).
  • Add C-terminal His8-tag after CBD.

Materials

BSA clone

  • BSA cDNA in pCMV-SPORT6
  • IMAGE clone ID # 8838824
  • E. coli DH10B TonA
  • glycerol stock frozen at -80°C

PCR primers

  • All primers from IDT
    • 100 nmol scale
    • HPLC purified
  • BSA cloning
    • BSA-NheI-5'
5'- CCC ACA AAA CTA TGG CTA GCA AGT GGG TGA CT-3'
Tm = 64.4°C
MW = 9842.4 g/mol
19.3 nmol, 0.19 mg
    • BSA-SapI-3'
5'-TGG TTG GCT CTT CTA CGG GCT AAG GCT GT-3'
Tm = 66.0°C
MW = 8946.8 g/mol
16.9 nmol, 0.15 mg
  • His-tag cloning
    • ins_His_after_CBD
5'-CCT TGT GGC AGC TTC AAC ACC ACC ATC ATC ATC ACC ACC ACT GAC TGC AGG AAG GG-3'
Tm = 72.1°C
MW = 17083.1 g/mol
8.0 nmol, 0.14 mg
    • Rins_His_after_CBD
5'-CCC TTC CTG CAG TCA GTG GTG GTG ATG ATG ATG GTG GTG TTG AAG CTG CCA CAA GG-3'
Tm = 72.1°C
MW = 17398.3 g/mol
7.4 nmol, 0.13 mg

pTXB1

  • From NEB
  • 200 μg/mL


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