Haynes Lab:Notebook/Engineering PC-TFs/2012/02/16: Difference between revisions
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==Ligation/Assembly== | ==Ligation/Assembly== | ||
* | * Weigh gel slices. | ||
* Add three times amount of volume to each tube of gel (600 µL) | * Add three times amount of volume to each tube of gel (600 µL) | ||
* Incubate at 55°C for 5-10 minutes. | * Incubate at 55°C for 5-10 minutes. | ||
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Results: | Results: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:# | | align="center" style="background:#848482;"|'''Molarity of DNA Fragments''' | ||
|- | |- | ||
| 1. KAH01 96.477 ng/µL | | 1. KAH01 96.477 ng/µL | ||
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*For all ligations, 25 ng of DNA vector = .3 µL | *For all ligations, 25 ng of DNA vector = .3 µL | ||
*There is a 2X molar ratio of insert:vector. | *There is a 2X molar ratio of insert:vector | ||
{| {{table}} | |||
|align="center" style="background:#f0f0f1;"|'''µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)''' | |||
|} | |||
Assembly Scheme: | |||
Number beside vector and insert is number of base pairs | |||
*1. hPCD (186) + KAH204 (2328) | |||
*2. hPCD (186) + KAH205 (1251) | |||
*3. hPCD (186) + KAH206 (1551) | |||
*4. hPCD (186) + KAH225 (1251) | |||
*5. hPCD (186) only | |||
{| {{table}} | |||
| align="center" style="background:#800000;"|'''''' | |||
| align="center" style="background:#FFB300;"|'''1''' | |||
| align="center" style="background:#800000;"|'''2''' | |||
| align="center" style="background:#FFB300;"|'''3''' | |||
| align="center" style="background:#800000;"|'''4''' | |||
| align="center" style="background:#FFB300;"|'''5''' | |||
|- | |||
| DNA Insert||1.3||0.4||0.5||0.3||----------- | |||
|- | |||
| DNA Vector||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||3.5||3.7||4 | |||
|- | |||
| ||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 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. | |||
*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 | |||
|} | |||
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Revision as of 08:48, 6 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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||
Ligation/Assembly
Measure concentration of each fragment:
Results:
Assembly Scheme: Number beside vector and insert is number of base pairs
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