Haynes Lab:Notebook/Engineering PC-TFs/2012/04/05: Difference between revisions
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|style="background-color: # | |style="background-color: #800000"|[[Image:HAYNESLAB3.jpeg|128px]]<span style="font-size:22px;"><span style="color:#FFCC00"> Engineering PC-TFs</span> | ||
|style="background-color: # | |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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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== | ==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 | |||
{| {{table}} | |||
| align="center" style="background:#0095B6;"|'''''' | |||
| align="center" style="background:#0095B6;"|'''1''' | |||
| align="center" style="background:#0095B6;"|'''2''' | |||
| align="center" style="background:#0095B6;"|'''3''' | |||
| align="center" style="background:#0095B6;"|'''4''' | |||
| align="center" style="background:#0095B6;"|'''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: | |||
{| {{table}} | |||
|align="center" style="background:#f0f0f1;"|'''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 | |||
|} | |||
[[Image:flyligation01.jpg|400px|thumb|left|Ligation Results]] | |||
10:1 ratio with negative control | |||
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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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||
4/5/12
Number beside vector and insert is number of base pairs
10:1 ratio with negative control |