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

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 159: Line 159:
::→ 37C O/N
::→ 37C O/N


==Categories==
[[Category:Escherichia coli]]
[[Category:Escherichia coli]]
[[Category:Escherichia coli/Vectors]]
[[Category:Vectors]]


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 08:38, 25 May 2011

AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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.

Special 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

Experimental Plan

  1. Miniprep BSA clone
    1. streak clone on LB+Amp100 plates - today
      1. O/N @ 37°C
    2. inoculate 5 mL LB+Amp100 - tomorrow
      1. O/N @ 37°C
    3. Miniprep - Thurs
  2. Dilute primers to 100 μM with sterile H2O
    1. Dilute BSA cloning primers to 4 μM for subcloning PCR
    2. Dilute His-tag primers to 12.5 ng/μL for QuikChange
  3. BSA PCR - Thurs afternoon
Basic PCR protocol
Tube sterile H2O Pfu Buffer Template 5'primer 3'primer dNTPs Pfu Turbo wax
(10X) (10 μg/mL) (4 μM) (4 μM) (10 mM ea) (2.5 U/μL)
A 72 μL 10 μL 5 μL 5 μL 5 μL 2 μL 1 μL 50 μL
B 73 μL 10 μL 5 μL 5 μL 5 μL 2 μL 0 μL 50 μL
  • Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
  1. 5' @ 95°C
  2. 30" @ 95°C
  3. 2' @ 60°C
  4. 2' @ 78°C
  5. repeat steps 2-4 x 29 more times
  6. 5' @ 78°C
  1. analytical minigel - Fri afternoon
  2. purify by spin column - Fri afternoon
  3. QuikChange of pTXB1 (when plasmid arrives)
    4. Basic QuikChange protocol
    Tube sterile H2O Pfu Buffer Template For primer Rev primer dNTPs Pfu Turbo wax
    (10X) (10 μg/mL) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
    A 12 μL 10 μL 10 μL 10 μL 5 μL 2 μL 1 μL 50 μL
    B 13 μL 10 μL 10 μL 10 μL 5 μL 2 μL 0 μL 50 μL
    • Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
    1. 30" @ 95°C
    2. 30" @ 95°C
    3. 1' @ 55°C
    4. 8.5' @ 68°C
    5. repeat steps 2-4 x 17 more times
    6. 10' @ 68°C
  4. DpnI digestion
    1. add 1μL DpnI to PCR tube
    2. 1h @ 37°C
  5. analytical minigel of pTXB1 PCR
  6. transform into DH10B
    • May need to make competent cells
  7. Miniprep
  8. sequence
  9. Sequential double-digest
    1. NheI digest
      2. NheI digest protocol
      Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL)
      plasmid 34.5 μL 5 μL 0.5 μL 5 μL of 100-500 μg/mL 5μL
      insert 24.5 μL 5 μL 0.5 μL 15 μL from purified PCR reaction 5μL
      1. 2h @ 37°C
      2. 20' @ 65°C
    2. SapI digest
      3. SapI digest protocol
      Tube sterile H2O 10X NEB4 DNA SapI (2 kU/mL)
      plasmid 30 μL 5 μL 10 μL of NheI digest 5μL
      insert 30 μL 5 μL 10 μL of NheI digest 5μL
      1. 2h @ 37°C
      2. spin-column purify
    3. Phosphatase vector
    4. gel purify
  10. ligate
  11. transform DH10B
  12. miniprep
  13. sequence
  14. express

Today's benchwork

  1. Streak out BSA clone on LBAmp100
    • Plates made by TF and KW yesterday
→ 37C O/N
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Kathryn_Muratore/Notebook/AU_CHEM-570_lab_prep/2011/05/24&oldid=511065"