IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/17

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dspB Track

Re-PCR with His tag off the genome

Protocol: SOP

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP0.5
sdH2O9.15
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA3
10uM FW primer (upHis)2
10uM RE primer (dw)2
Total25

Tubes: His, W1 (with primers), W2 (without primers)

PCR Cycles:

  • 98C @ 30sec
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
  • 72C @ 10 min
  • 10C @ hold

Start: 3:18pm
End: 4:19pm

Gel verification for re-PCR (above)

  • Protocol: biobrick "digest" protocol

Gel orientation:

Gel orientation
His100bp ladderW1W2

Machine conditions: 0.5x TBE buffer, 100V, 45min Results:
File:100917 dspB 35.tif

PCR Purification on his-PCR product

  • Elute with 30uL
  • [His] = 23.5ng/uL

Restriction Digest on his PCR Product

Restriction Digest
REAGENTS1 RXN VOLUME (uL)
Buffer 22
BSA0.2
DNA10
ddH205.8
Total20

Enzymes: Use XbalI & PstI (1uL each) DNA: his PCR product

Ligation

  • Ratio: 3:1

Calculations
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)}
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 3 \times \frac{1200}{2400} \times = ng}
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }

  • Where x = concentration of insert
  • 0.7uL vector: 17uL insert
  • Protocol: "Ligate" protocol from biobrick
  • Ligase: add 1uL; T4 ligase buffer: add 2uL

[cont. from yesterday]Gel Extraction of His cut at X/P

  • Weight: 0.1548g
  • Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)
  • 10uL of NCC (1288.3ng/uL) to digest = 1288ng
  • 12883ng of NCC in 20uL = 644.15ng/uL
  • Assume half of this: 322.08ng/uL
  • For EB buffer, add 25uL

Nanodrop:

  • [XP dspB extraction] = 6.9ng/uL (25uL in total)

Ligation

  • Ratio: 3:1

Calculations
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)}
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 3 \times \frac{1200}{2400} \times = ng}
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }

  • Where x = concentration of insert
  • Rafael's suggestion: for pre-gel extract, don't use the [] from the nanodrop (7.0ng/uL). Instead, take half the concentration of what was in the digest, in case the agarose is preventing an accurate reading on the nanodrop.
  • 2uL of vector: 5.2uL of His
  • Protocol: "Ligate" protocol from biobrick
  • Tube: XP dspB gel extract + pre gel extract -> pre-xpdspB lig
  • Incubate at RT for 20min

Transformation

  • Use 5uL of ligation mix
  • In 37C incubator at 10:00pm