User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/05/24

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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.

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

Experimental Plan

 * 1)  Miniprep BSA clone
 * 2) streak clone on LB+Amp100 plates - today
 * 3) O/N @ 37°C
 * 4) inoculate 5 mL LB+Amp100 - tomorrow
 * 5) O/N @ 37°C
 * 6) Miniprep - Thurs
 * 7) Dilute primers to 100 μM with sterile H2O
 * 8) Dilute BSA cloning primers to 4 μM for subcloning PCR
 * 9) Dilute His-tag primers to 12.5 ng/μL for QuikChange
 * 10) BSA  PCR - Thurs afternoon
 * 11) * Tanneal = 60°C
 * 12) analytical minigel - Fri afternoon
 * 13) purify by spin column - Fri afternoon
 * 14) QuikChange of pTXB1 (when plasmid arrives)
 * 15) DpnI digestion
 * 16) add 1μL DpnI to PCR tube
 * 17) 1h @ 37°C
 * 18) analytical minigel of pTXB1 PCR
 * 19) transform into DH10B
 * 20) * May need to make competent cells
 * 21) Miniprep
 * 22) sequence
 * 23) Sequential double-digest
 * 24) NheI digest
 * 25) SapI digest
 * 26) Phosphatase vector
 * 27) gel purify
 * 28) ligate
 * 29) transform DH10B
 * 30) miniprep
 * 31) sequence
 * 32) express

Today's benchwork

 * 1) Streak out BSA clone on  LBAmp100
 * 2) * Plates made by TF and KW yesterday
 * &rarr; 37C O/N


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