Difference between revisions of "Team:BNU-China/Protocol"

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                     </figure>
 
                     </figure>
 
                     <h3>4) Fusion PCR</h3>
 
                     <h3>4) Fusion PCR</h3>
−
                     <p>1. basic PCR</br>2. use the PCR product of step 1 as template to do PCR</br>3. use the PCR product of step 2 as template to do PCR, but first five cycles don’t add primer, add primer at the sixth cycle and continue PCR.</p>
+
                     <p>1. Basic PCR</br>2. Use the PCR product of step 1 as template to do PCR</br>3. Use the PCR product of step 2 as template to do PCR, but first five cycles don't add primer, add primer at the sixth cycle and continue PCR.</p>
 
                     <p>The system of step 2:</br>
 
                     <p>The system of step 2:</br>
 
H2O 21μL</br>
 
H2O 21μL</br>
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Template2 1μL
 
Template2 1μL
 
</p>
 
</p>
 +
                    <h2>2. Plasmid concentration</h2>
 +
                    <p>1. 1. Dilute collected DNA to 400μl. (If the plasmid is eluted by ddH2O, then it is diluted by ddH2O as well; If the plasmid is eluted by elution buffer, then it is diluted by elution buffer as well)</p>
 
                      
 
                      
 
                      
 
                      

Revision as of 18:12, 17 October 2018

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Protocol

1. PCR system

1) 20μL system is used for verification

Taq:

Super mix:

2) 50μL system for PCR target genes

Pfu mix:

DNA: plasmid 50ng; genome 100~200ng

3) 20μL PCR system for microbes

Super mix:

Bacteria (after picking on the board, back up on the new board and rinse it in the system that has been prepared)

4) Fusion PCR

1. Basic PCR
2. Use the PCR product of step 1 as template to do PCR
3. Use the PCR product of step 2 as template to do PCR, but first five cycles don't add primer, add primer at the sixth cycle and continue PCR.

The system of step 2:
H2O 21μL
2x primeSTAR 25μL
R+F-Primer 2 μL
Template1 1μL
Template2 1μL

2. Plasmid concentration

1. 1. Dilute collected DNA to 400μl. (If the plasmid is eluted by ddH2O, then it is diluted by ddH2O as well; If the plasmid is eluted by elution buffer, then it is diluted by elution buffer as well)