Haynes Lab:Notebook/Engineering PC-TFs/2012/03/01: Difference between revisions
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(5 intermediate revisions by the same user not shown) | |||
Line 10: | Line 10: | ||
Assembly Scheme: | Assembly Scheme: | ||
Number beside vector and insert is number of base pairs | Number beside vector and insert is number of base pairs | ||
1. | *1. hPCD (186) + KAH204 (2328) | ||
2. | *2. hPCD (186) + KAH205 (1251) | ||
3. | *3. hPCD (186) + KAH206 (1551) | ||
4. | *4. hPCD (186) + KAH225 (1251) | ||
5. | *5. hPCD (186) only | ||
{| {{table}} | {| {{table}} | ||
Line 33: | Line 32: | ||
| Lign Buffer (2x)||5||5||5||5||5 | | Lign Buffer (2x)||5||5||5||5||5 | ||
|- | |- | ||
| dH2O||2.4||3.3||3.5||3 | | 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 | |||
*Add 30 μL of DH5α Turbo cells to DNA + dH2O | |||
*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 | |||
|} | |||
Template Table | |||
{| {{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||--||--||--||--||----------- | |||
|- | |||
| DNA Vector||--||--||--||--||-- | |||
|- | |||
| T4 Ligase||1||1||1||1||1 | |||
|- | |||
| Lign Buffer (2x)||5||5||5||5||5 | |||
|- | |||
| dH2O||--||--||--||--||-- | |||
|- | |- | ||
| ||10 µL||10 µL||10 µL||10 µL||10 µL | | ||10 µL||10 µL||10 µL||10 µL||10 µL |
Revision as of 11:34, 5 September 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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SummaryAssembly Scheme: Number beside vector and insert is number of base pairs
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