Haynes Lab:Notebook/Engineering PC-TFs/2012/02/16: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 47: Line 47:
{| {{table}}
{| {{table}}
|align="center" style="background:#f0f0f1;"|'''µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)'''
|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 (3386) + KAH204 (2328)
2. hPCD (3386) + KAH205 (1251)
3. hPCD (3386) + KAH206 (1551)
4. hPCD (3386) + KAH225 (1251)
5. hPCD (3386) only


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 19:27, 17 February 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>

Ligation/Assembly

  • Weight gel slices.
  • Add three times amount of volume to each tube of gel (600 µL)
  • Incubate at 55°C for 5-10 minutes.
  • Centrifuge for a minute at 17,000 x g.
  • Add 20 µL of wash buffer.
  • Don't dump supernatent and centrifuge for one minute again.
  • Place spin columns in 1.5 mL tubes.

Measure concentration of each fragment:

  • Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
  • Go to Nucleic Acid Quantification.
  • Pipette 2 µL of each DNA sequence into Take 3 plate.
  • Place plate in EPOCH plate reader.

Results:

Molarity of DNA Fragments
1. KAH01 96.477 ng/µL
2. KAH03 122.149 ng/µL
3. KAH04 80.182 ng/µL
4. KAH204 27.054 ng/µL
5. KAH205 47.105 ng/µL
6. KAH206 45.217 ng/µL
7. KAH225 66.047 ng/µL


  • For all ligations, 25 ng of DNA vector = .3 µL
  • There is a 2X molar ratio of insert:vector
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 (3386) + KAH204 (2328) 2. hPCD (3386) + KAH205 (1251) 3. hPCD (3386) + KAH206 (1551) 4. hPCD (3386) + KAH225 (1251) 5. hPCD (3386) only



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2012/02/16&oldid=585815"