Team:TU Delft/24 June 2010 content

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(New page: Today we colony PCR'd some of the transformants presumably containing the BioBricks we will be using for the rest of the Summer. We hope for the best and to have the results in over 1 hour...)
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Today we colony PCR'd some of the transformants presumably containing the BioBricks we will be using for the rest of the Summer. We hope for the best and to have the results in over 1 hour!
Today we colony PCR'd some of the transformants presumably containing the BioBricks we will be using for the rest of the Summer. We hope for the best and to have the results in over 1 hour!
 +
The protocol followed for colony PCR is as follows:
The protocol followed for colony PCR is as follows:
-
-  
+
 
 +
 
 +
''Materials:''
 +
 
 +
- Taq PCR Master Mix (Qiagen) is a premixed solution containing Taq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP.
 +
 
 +
- Primer solutions 5 mol/mL
 +
 
 +
 
 +
''Protocol:''
 +
 
 +
First make sure that there is a PCR machine available for you.
 +
Take the solutions from the freezer and thaw on ice.
 +
Preparation of reaction mixture:
 +
 
 +
1. Gently vortex and briefly centrifuge all solutions after thawing
 +
 
 +
2. Keep solutions on ice
 +
 
 +
3. Make a pre-mix for the amount of colonies (to be analyzed) + 1, add to an Eppendorf tube:
 +
 
 +
1× pre-mix
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|Component
 +
|Sample
 +
|-
 +
|Taq PCR Master Mix (Qiagen)
 +
|25 μL
 +
|-
 +
|G0010
 +
|3 μL
 +
|-
 +
|G0011
 +
|3 μL
 +
|-
 +
|H20
 +
|29 μL
 +
|}
 +
 
 +
 
 +
4. Add 50 μL of pre-mix to each PCR tube.
 +
 
 +
5. Prick a sterile toothpick into a colony, dip it into a PCR tube, put it into a 15 mL culture tube containing 5 mL LB medium + antibiotics and then grow overnight for mini-prep cultures and -80 °C stocks. Repeat this for all the colonies. Incubate the mini-prep cultures at 37 °C.
 +
 
 +
6. Keep everything ice cold until you put the tubes in the preheated PCR machine
 +
 
 +
 
 +
PCR program
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|Step
 +
|Annealing Temperature
 +
|Time, min:sec
 +
|Number of cycles
 +
|-
 +
|Initial denaturation
 +
|94 °C
 +
|2:00
 +
|1
 +
|-
 +
|Denaturation
 +
|94 °C
 +
|1:00
 +
|30
 +
|-
 +
|Annealing
 +
|x °C *
 +
|0:45
 +
|30
 +
|-
 +
|Extension
 +
|72 °C
 +
|1:00
 +
|30
 +
|-
 +
|Extension
 +
|72 °C
 +
|5:00
 +
|1
 +
|}
 +
 
 +
* Calculate the optimal annealing temperature = 3x G/C + 2x A/T
 +
 
The ligation products of yesterday were transformed into our awesomely competent cells.
The ligation products of yesterday were transformed into our awesomely competent cells.

Revision as of 14:05, 24 June 2010

Today we colony PCR'd some of the transformants presumably containing the BioBricks we will be using for the rest of the Summer. We hope for the best and to have the results in over 1 hour!


The protocol followed for colony PCR is as follows:


Materials:

- Taq PCR Master Mix (Qiagen) is a premixed solution containing Taq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP.

- Primer solutions 5 mol/mL


Protocol:

First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw on ice. Preparation of reaction mixture:

1. Gently vortex and briefly centrifuge all solutions after thawing

2. Keep solutions on ice

3. Make a pre-mix for the amount of colonies (to be analyzed) + 1, add to an Eppendorf tube:

1× pre-mix

Component Sample
Taq PCR Master Mix (Qiagen) 25 μL
G0010 3 μL
G0011 3 μL
H20 29 μL


4. Add 50 μL of pre-mix to each PCR tube.

5. Prick a sterile toothpick into a colony, dip it into a PCR tube, put it into a 15 mL culture tube containing 5 mL LB medium + antibiotics and then grow overnight for mini-prep cultures and -80 °C stocks. Repeat this for all the colonies. Incubate the mini-prep cultures at 37 °C.

6. Keep everything ice cold until you put the tubes in the preheated PCR machine


PCR program

Step Annealing Temperature Time, min:sec Number of cycles
Initial denaturation 94 °C 2:00 1
Denaturation 94 °C 1:00 30
Annealing x °C * 0:45 30
Extension 72 °C 1:00 30
Extension 72 °C 5:00 1
  • Calculate the optimal annealing temperature = 3x G/C + 2x A/T


The ligation products of yesterday were transformed into our awesomely competent cells.

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