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

From OpenWetWare
Jump to navigationJump to search
Line 90: Line 90:
|align="center" style="background:#f0f0f1;"|'''Transformation Process'''
|align="center" style="background:#f0f0f1;"|'''Transformation Process'''


Warm five 100 μg/mL Amp agar plates at 37 °C
*Warm five 100 μg/mL Amp agar plates at 37 °C
Thaw fresh tube of DH5α Turbo cells on ice
*Thaw fresh tube of DH5α Turbo cells on ice
Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O
*Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O
Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes
*Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes
Add 30 μL of DH5α Turbo cells to DNA + dH2O
*Add 30 μL of DH5α Turbo cells to DNA + dH2O
Include #5 tube, water only, no DNA (negative control)
*Include #5 tube, water only, no DNA (negative control)
Incubate cells + DNA on ice for 5 min.
*Incubate cells + DNA on ice for 5 min.
Label pre-warmed plates
*Label pre-warmed plates
Transfer cells + DNA onto agar
*Transfer cells + DNA onto agar
Add 10 - 15 sterile glass beads, shake, discard beads
*Add 10 - 15 sterile glass beads, shake, discard beads
Incubate plates at 37 °C overnight
*Incubate plates at 37 °C overnight
|}
|}


Revision as of 19:43, 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
' 1 2 3 4 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
30 µL 30 µL 30 µL 30 µL 30 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
  • Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O
  • Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes
  • 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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2012/02/16&oldid=585824"