Team:TU Delft/24 June 2010 content

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(Characterization of Anderson RBS sequences)
 
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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!
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=Lab work=
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==Characterization of Anderson RBS sequences==
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The ligation products of [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=23_June_2010 yesterday] were transformed into Top10 competent cells.
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The protocol followed for colony PCR is as follows:
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=World Championship=
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The final group match for the Netherlands in the World Cup. A big screen was set up in the botanical garden next to the BT building of TU Delft to watch the game with the complete department 
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<html>
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''Materials:''
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<a href="http://2010.igem.org/wiki/images/5/56/Vuvuzela.ogg" target="vuvuframe">Support Holland!</a>
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<font size="1"><a href="about:blank" target="vuvuframe">Stop sound</a></font>
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- 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.
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<iframe name="vuvuframe" src="about:blank" width="1" height="1" border="0" style="border:none;"></iframe>
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</html>
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- Primer solutions 5 mol/mL
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''Protocol:''
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First make sure that there is a PCR machine available for you.
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Take the solutions from the freezer and thaw on ice.
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Preparation of reaction mixture:
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1. Gently vortex and briefly centrifuge all solutions after thawing
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2. Keep solutions on ice
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3. Make a pre-mix for the amount of colonies (to be analyzed) + 1, add to an Eppendorf tube:
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1× pre-mix
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|Component
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|Sample
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|Taq PCR Master Mix (Qiagen)
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|25 μL
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|G0010
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|3 μL
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|G0011
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|3 μL
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|H20
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|29 μL
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|}
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4. Add 50 μL of pre-mix to each PCR tube.
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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.
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6. Keep everything ice cold until you put the tubes in the preheated PCR machine
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PCR program
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|Step
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|Annealing Temperature
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|Time, min:sec
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|Number of cycles
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|Initial denaturation
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|94 °C
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|2:00
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|1
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|-
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|Denaturation
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|94 °C
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|1:00
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|30
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|-
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|Annealing
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|x °C *
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|0:45
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|30
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|Extension
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|72 °C
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|1:00
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|30
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|Extension
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|72 °C
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|5:00
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|1
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|}
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* Calculate the optimal annealing temperature = 3x G/C + 2x A/T
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The ligation products of yesterday were transformed into our awesomely competent cells.
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Latest revision as of 12:29, 3 August 2010

Lab work

Characterization of Anderson RBS sequences

The ligation products of yesterday were transformed into Top10 competent cells.

World Championship

The final group match for the Netherlands in the World Cup. A big screen was set up in the botanical garden next to the BT building of TU Delft to watch the game with the complete department

Support Holland! Stop sound