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

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==Ligation/Assembly==
==Ligation/Assembly==
* Weight gel slices.
* 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:#f0f0f0;"|'''Molarity of DNA Fragments'''
| align="center" style="background:#848482;"|'''Molarity of DNA Fragments'''
|-
|-
| 1. KAH01    96.477  ng/µL
| 1. KAH01    96.477  ng/µL
Line 52: Line 52:
Number beside vector and insert is number of base pairs
Number beside vector and insert is number of base pairs


*1. hPCD (3386) + KAH204 (2328)
*1. hPCD (186) + KAH204 (2328)
*2. hPCD (3386) + KAH205 (1251)
*2. hPCD (186) + KAH205 (1251)
*3. hPCD (3386) + KAH206 (1551)
*3. hPCD (186) + KAH206 (1551)
*4. hPCD (3386) + KAH225 (1251)
*4. hPCD (186) + KAH225 (1251)
*5. hPCD (3386) only
*5. hPCD (186) only


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#800000;"|''''''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#FFB300;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#800000;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#FFB300;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#800000;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#FFB300;"|'''5'''
|-
|-
| DNA Insert||1.3||0.4||0.5||0.3||-----------
| DNA Insert||1.3||0.4||0.5||0.3||-----------
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| dH2O||2.4||3.3||3.5||3.7||4
| dH2O||2.4||3.3||3.5||3.7||4
|-
|-
| ||30 µL||30 µL||30 µL||30 µL||30 µL
| ||10 µL||10 µL||10 µL||10 µL||10 µL
|-
|-
|  
|  
|}
|}
* In a .5 mL tube, pipette the ligation buffer first.
* In a .5 mL tube, pipette the ligation buffer first.
* Then water, DNA insert, vector, and T4 ligase.
* Then water, DNA insert, vector, and T4 ligase.
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|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 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
*Add 30 μL of DH5α Turbo cells to DNA.
*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.
*Incubate cells + DNA on ice for 5 min.
*Label pre-warmed plates
*Label pre-warmed plates

Revision as of 08:48, 6 September 2012

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Ligation/Assembly

  • Weigh 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 (186) + KAH204 (2328)
  • 2. hPCD (186) + KAH205 (1251)
  • 3. hPCD (186) + KAH206 (1551)
  • 4. hPCD (186) + KAH225 (1251)
  • 5. hPCD (186) 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
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:


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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