IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-13: Difference between revisions

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Each pot should have:
Each pot should have:
==PCR Reactions==
 
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''REACTION A'''
| align="center" style="background:#f0f0f0;"|'''REACTION A'''
| align="center" style="background:#f0f0f0;"|'''REACTION B'''
| align="center" style="background:#f0f0f0;"|'''REACTION C'''
| align="center" style="background:#f0f0f0;"|'''REACTION D'''
| align="center" style="background:#f0f0f0;"|'''REACTION E'''
|-
|-
| 2 mM [Mg] || 4 mM [Mg] || 6 mM [Mg]  
| 2 mM [Mg]
|-
|-
| 10 μL template || 10 μL template || 10 μL template || 5uL template || 1 uL template
| 10 μL template  
|-
|-
| 10 μL 10x buffer || 10 μL 10x buffer || 10 μL 10x buffer || 5 uL 10x buffer || 5 uL 10x buffer
| 10 μL 10x buffer
|-
|-
| 2 μL 10 mM dNTP || 2 μL 10 mM dNTP || 2 μL 10 mM dNTP || 1 uL 10mM dNTP
| 2 μL 10 mM dNTP  
|-
|-
| 2 μL Primer 1 || 2 μL Primer 1 || 2 μL Primer 1 || 1 uL Primer 1 || 1 uL Primer 2
| 2 μL Primer 1  
|-
|-
| 2 μL Primer 2 || 2 μL Primer 2 || 2 μL Primer 2 || 1 uL Primer 2 || 1 uL Primer 2
| 2 μL Primer 2
|-
|-
| 1 μL Taq || 1 μL Taq || 1 μL Taq || 0.5 uL Taq || 0.5 uL Taq
| 1 μL Taq  
|-
|-
| 73 μL dH<sub>2</sub>0 || 71 μL dH<sub>2</sub>0 || 69 μL dH<sub>2</sub>0 || 37 μL dH<sub>2</sub>0 || 41 μL dH<sub>2</sub>0  
| 73 μL dH<sub>2</sub>0  
|-
|-
| - || 2 μL 100 mM Mg || 4 μL 100 mM Mg
|  
|  
|  
|}
|}

Revision as of 10:31, 13 July 2006



Incubator status

  • Removed
    • ...
Click here for a map of the lower shelf
(2006-07-13)

Experimental Results

The plates grown from yesterday (2nd Topo&Transformation) show the following:

Positive Control: Many colonies

Negative Control: No colonies

Tranformed Plate: One in the middle, three+ on the edge of the plate.

Site-Specific Mutagenesis PCR

Refer to the image for details; this is the "crossover pcr" step.

Refer to this for PCR overview

For this step, we are going to create 4 PCR constructs with primers which will create the site mutation we want to eliminate the restriction sites.

The 4 sites are:

    1. kABC_3_9
    2. kABC_10_7
    3. kABC_8_5
    4. kABC_6_4

Where:

  • 3=crossF
  • 4=crossR
  • 5=pst1R
  • 6=pst1F
  • 7=pst2R
  • 8=pst2F
  • 9=eco1R
  • 10=eco1F

and:

  • the kABC_x_y signifies the sequence bounded by primers 'x' and 'y'

The cycle for this PCR is: 

 *#95@15'
 *#94@30"
 *#56@30"
 *#72@3'30"
 *#Cycle to step 2, 40x
 *#72@5"
 *#4@forever

Each pot should have:

REACTION A
2 mM [Mg]
10 μL template
10 μL 10x buffer
2 μL 10 mM dNTP
2 μL Primer 1
2 μL Primer 2
1 μL Taq
73 μL dH20

Future Experiment Planning

More detailed plans for our future cyanobacteria work:
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