Difference between revisions of "Golden Gate Assembly for Bio113"

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(Created page with '<center>'''Protocol to insert new promoter made into pSB1A2-BR that contains [http://partsregistry.org/Part:BBa_J100091 BBa_J100091]'''</center><br> <br> ==Starting with Boiled…')
 
 
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<center>'''Protocol to insert new promoter made into pSB1A2-BR that contains [http://partsregistry.org/Part:BBa_J100091 BBa_J100091]'''</center><br>  <br>
 
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==Starting with Boiled then Cooled Oligos==
 
==Starting with Boiled then Cooled Oligos==
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# You should have already used the [http://gcat.davidson.edu/iGem10/index.html Oligator] to design your oligos for your dsDNA promoter. <br>
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# You should have already used the [http://gcat.davidson.edu/iGem10/index.html Oligator] to design your oligos for your dsDNA control element. <br>
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# Go to the [http://gcat.davidson.edu/SynBio12/ Loligator web page] to determine how to prepare your oligos to add to the solution described below. <br>
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# You should have already calculated how to dilute your boiled and cooled oligos from 5 µM to 40 nM. <br>
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If you don't have time to ethanol precipitate and quantify your DNA, you can use this shortcut:
 
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* 1 µL cooled oligos.
 
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* 99 µL water to make a 100X dilution.
 
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* Use 1 µL of the 100X diluted oligos and add to the 9 µL described below.
 
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<br>
 
  
 
==Protocol Details for GGA==
 
==Protocol Details for GGA==
  
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# Get '''two''' new, small microfuge tubes designed to fit into the PCR machine.
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# You will be given two tubes. One tube will be labeled "X" for eXperimental DNA, the other "N" for the negative control that contains plasmid only. Put your initials on the tubes and return the tubes to ice. These tubes will already contain 9 µL of the GGA Mixture.
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# One tube will be labeled "GGA ligation", the other "GGA plasmid only". Put your initials on the tubes. These tubes will already contain 9 µL of the GGA Mixture.
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# Add 1 µL '''diluted''' oligos cooled overnight to the tube labeled '''X''' for the experimental DNA.
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# Add 1 µL '''diluted''' oligos cooled overnight to the tube labeled GGA ligation.
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# Add 1 µL '''water''' to the tube labeled '''N''' for the negative control"
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# Add 1 µL water to the tube labeled "GGA plasmid only"
 
  
 
'''GGA mixture contains:'''<br>
 
'''GGA mixture contains:'''<br>
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1 µL (50 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100091 part J100091]<br>  
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* 1 µL (40 nM) plasmid containing your receiving plasmid (pClone)<br>  
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5 µL dH<sub>2</sub>O <br>  
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* 6 µL dH<sub>2</sub>O <br>  
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1 µL 10X Promega Ligase Buffer<br>  
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* 1 µL 10X Promega Ligase Buffer<br>  
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1 µL 500 mM KOAc<br>
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* 0.5 µL Bsa I high fidelity (HFv2) restriction enzyme<br>  
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0.5 µL Bsa I high fidelity (HF) restriction enzyme<br>  
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* <u>0.5 µL T4 DNA ligase from Promega</u><br>  
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<u>0.5 µL T4 DNA Ligase</u><br>  
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* 9 µL final volume <br> <br>  
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9 µL final volume <br> <br>  
 
  
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Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br>  
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Turn on the thermocycler. Put '''both''' of your tubes into the machine. <br>  
 
Program it for the following cylces: <br>  
 
Program it for the following cylces: <br>  
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* 20 cycles of 37C for 1 minute/16C for 1 minute <br>  
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* 20 cycles of 37C for 1.5 minute and 16C for 1.5 minute <br>  
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* 1 cycle of 37C for 15 minutes <br>
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* 37C for 3 minutes<br>
 
* 22C holding temperature <br>  
 
* 22C holding temperature <br>  
 +
* heated lid on
 +
 
This DNA ligation is ready for transformation. <br> <br>  
 
This DNA ligation is ready for transformation. <br> <br>  
  
 
You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
 
You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
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==Transformations after GGA==
 
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You want to do 3 transformations:<br>
 
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# a positive control that contains known [http://partsregistry.org/Part:BBa_K315008 promoter + RBS + RFP] to be used for comparison RFP expression. <br>
 
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# your experimental GGA ligation<br>
 
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# your negative control "GGA plasmid only" ligation.<br><br>
 
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Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. <br>
 
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You will want to perform a transformation positive control using [http://partsregistry.org/Part:BBa_K315008 K315008] which contains pLacI+RBS+RFP in plasmid pSB1A2. <br>  <br>
 
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Use between 20 - 50 µL of Zippy competent cells into each of three 1.5 mL tubes labeled appropriately for each of the three [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformations] listed above. Plate all three transformations on LB amp plates.<br>  <br>
 
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==PCR Verification of Successful GGA==
 
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If you want to PCR verify that GGA has happened, you can use colony PCR and analyze the product by gel electrophoresis (1.7% agarose gel).
 
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# Use negative control colonies as template to see the MW of the PCR product when TT is still in the plasmid and no GGA has occurred. Run one lane of this negative control PCR product for each row on your gel.
 
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# Use these two primers:
 
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* Forward = 5’ GAATTCGCGGCCGCTTCTAG  3’
 
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* Reverse = 5’ TTTGATAACATCTTCGGAGG  3’
 
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* PCR product with the original TT still in place is 251 bp
 
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* size of TT that should be removed by GGA is 107 bp
 
  
  
 
This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by Annie Wacker and Malcolm Campbell.
 
This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by Annie Wacker and Malcolm Campbell.

Latest revision as of 14:02, 5 February 2020

Starting with Boiled then Cooled Oligos

  1. You should have already used the Oligator to design your oligos for your dsDNA control element.
  2. You should have already calculated how to dilute your boiled and cooled oligos from 5 µM to 40 nM.

Protocol Details for GGA

  1. You will be given two tubes. One tube will be labeled "X" for eXperimental DNA, the other "N" for the negative control that contains plasmid only. Put your initials on the tubes and return the tubes to ice. These tubes will already contain 9 µL of the GGA Mixture.
  2. Add 1 µL diluted oligos cooled overnight to the tube labeled X for the experimental DNA.
  3. Add 1 µL water to the tube labeled N for the negative control"

GGA mixture contains:

  • 1 µL (40 nM) plasmid containing your receiving plasmid (pClone)
  • 6 µL dH2O
  • 1 µL 10X Promega Ligase Buffer
  • 0.5 µL Bsa I high fidelity (HFv2) restriction enzyme
  • 0.5 µL T4 DNA ligase from Promega
  • 9 µL final volume

Turn on the thermocycler. Put both of your tubes into the machine.
Program it for the following cylces:

  • 20 cycles of 37C for 1.5 minute and 16C for 1.5 minute
  • 37C for 3 minutes
  • 22C holding temperature
  • heated lid on

This DNA ligation is ready for transformation.

You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.


This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by Annie Wacker and Malcolm Campbell.

Retrieved from "https://gcat.davidson.edu/GcatWiki/index.php?title=Golden_Gate_Assembly_for_Bio113&oldid=19282"