Haynes Lab:Notebook/Engineering PC-TFs/2012/04/05: Difference between revisions

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|style="background-color: #FFCC00"|[[Image:HAYNESLAB3.jpeg|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|style="background-color: #800000"|[[Image:HAYNESLAB3.jpeg|128px]]<span style="font-size:22px;"><span style="color:#FFCC00"> Engineering PC-TFs</span>
|style="background-color: #800000" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #FFCC00" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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* Ligation Scheme
* Ligation Scheme
Number beside vector and insert is number of base pairs
Number beside vector and insert is number of base pairs
*1. flyPCD (3386) + KAH204 (2328)
*1. flyPCD (186) + KAH204 (2328)
*2. flyPCD (3386) + KAH205 (1251)
*2. flyPCD (186) + KAH205 (1251)
*3. flyPCD (3386) + KAH206 (1551)
*3. flyPCD (186) + KAH206 (1551)
*4. flyPCD (3386) + KAH225 (1251)
*4. flyPCD (186) + KAH225 (1251)
*5. flyPCD (3386) only
*5. flyPCD (186) only


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Revision as of 11:30, 25 August 2012

Engineering PC-TFs <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

4/5/12

  • Ligation Scheme

Number beside vector and insert is number of base pairs

  • 1. flyPCD (186) + KAH204 (2328)
  • 2. flyPCD (186) + KAH205 (1251)
  • 3. flyPCD (186) + KAH206 (1551)
  • 4. flyPCD (186) + KAH225 (1251)
  • 5. flyPCD (186) only
' 1 2 3 4 5
DNA Insert KAH### 1.3 0.4 0.5 0.3 -----------
DNA Vector(flyPCD) 0.3 0.3 0.3 0.3 0.3
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O 2.4 3.3 3.2 3.4 3.6
10 µL 10 µL 10 µL 10 µL 10 µL
  • In a .5 mL tube, pipette the ligation buffer first.
  • Then water, DNA insert, vector, and T4 ligase.
  • Once completed, allow them to incubate for 10 minutes.
  • Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
  • Then follow transformation process:


Transformation Process
  • Warm five 100 μg/mL Amp agar plates at 37 °C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Include #5 tube, water only, no DNA (negative control)
  • Incubate cells + DNA on ice for 5 min.
  • Label pre-warmed plates
  • Transfer cells + DNA onto agar
  • Add 10 - 15 sterile glass beads, shake, discard beads
  • Incubate plates at 37 °C overnight
Ligation Results

10:1 ratio with negative control


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