Team:TU Darmstadt/Notebook/Methods/Colony PCR with Taq polymerase

From 2014.igem.org

(Difference between revisions)
Line 14: Line 14:
<div id="wikicontent" class="grid_19">
<div id="wikicontent" class="grid_19">
-
<!--TYPO3SEARCH_begin--><div id="c97" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Colony PCR with Taq polymerase</h1></div><div><p><b>Equipment:</b></p></div><div><p>- PCR machine</p></div><div></div><div><p><b>Chemicals &amp; consumables:</b></p></div><div><p>- Sterile Eppendorf Tubes</p></div><div><p>- LB-agar plate with appropriate antibiotic</p></div><div><p>- Primers (usually VF2 and VR)</p></div><div><p>- Sterile pipet tips</p><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; "><b style="border: 0px; margin: 0px; padding: 0px; ">Mixtures:&nbsp;</b>1x Reaction Mixture (25 µL)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 12,5 µL 2x Taq MM</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VF2 (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VR (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 1 µL of colony suspension</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- ddH<span style="border: 0px; margin: 0px; padding: 0px; font-size: 10px; line-height: 0; position: relative; vertical-align: baseline; bottom: -0.25em; ">2</span>O to 25 µL</p></div></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.</p></div><div><p>- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.</p></div><div><p>- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</p></div><div><p>Start the PCR using the following programm and 1X mix.</p></div><div><p>Run a gel to determine the product length (don't forget the positive control).</p></div></div><div id="c98" class="csc-default">
+
<!--TYPO3SEARCH_begin--><div id="c97" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Colony PCR with Taq polymerase</h1></div><div><p><b>Equipment:</b></p></div><div><p>- PCR machine</p></div><div></div><div><p><b>Chemicals &amp; consumables:</b></p></div><div><p>- Sterile Eppendorf Tubes</p></div><div><p>- LB-agar plate with appropriate antibiotic</p></div><div><p>- Primers (usually VF2 and VR)</p></div><div><p>- Sterile pipet tips</p><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; "><b style="border: 0px; margin: 0px; padding: 0px; ">Mixtures:&nbsp;</b>1x Reaction Mixture (25 µL)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 12,5 µL 2x Taq MM</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VF2 (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 0,5 µL VR (10 µM)</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- 1 µL of colony suspension</p></div><div style="border: 0px; margin: 0px; padding: 0px; font-size: 11px; color: rgb(102, 102, 102); font-family: Tahoma, sans-serif; line-height: 16.5px; "><p style="border: 0px; margin: 0px 0px 10px; padding: 0px; font-size: 13px; text-align: justify; ">- ddH<span style="border: 0px; margin: 0px; padding: 0px; font-size: 10px; line-height: 0; position: relative; vertical-align: baseline; bottom: -0.25em; ">2</span>O to 25 µL</p></div></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.</p></div><div><p>- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.</p></div><div><p>- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.</p></div><div><p>Start the PCR using the following programm and 1x mix.</p></div><div><p>Run a gel to determine the product length (don't forget the positive control).</p></div></div><div id="c98" class="csc-default">
<table class="table">
<table class="table">
<tr>
<tr>

Revision as of 18:41, 17 October 2014

Home


Colony PCR with Taq polymerase

Equipment:

- PCR machine

Chemicals & consumables:

- Sterile Eppendorf Tubes

- LB-agar plate with appropriate antibiotic

- Primers (usually VF2 and VR)

- Sterile pipet tips

Mixtures: 1x Reaction Mixture (25 µL)

- 12,5 µL 2x Taq MM

- 0,5 µL VF2 (10 µM)

- 0,5 µL VR (10 µM)

- 1 µL of colony suspension

- ddH2O to 25 µL

Procedure:

The colony PCR is a modified PCR programm employed to verifiy transformation success by amplifiying the insert or the vector construct used for transformation. This is necessary due to the fact, that a transformation with the empty vector may lead to antibiotic resistance.

- Pick one colony with a sterile tip and suspend in 10 µL of ddH2O.

- Inoculate tip with colony into tube. Pipet up and down to ensure all cells are transferred to tube.

Start the PCR using the following programm and 1x mix.

Run a gel to determine the product length (don't forget the positive control).

#   Temperature   Time
         
1   95°C   00:05:00
         
2   95°C   00:00:30
         
3   55°C   00:00:30
         
4   68°C   1 min/kbp
         
5   GO TO 2   REPEAT 30x
         
6   68°C   1.5 min/kbp
         
7   4°C   HOLD