Difference between revisions of "Golden Gate Assembly protocol"

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'''Protocol to insert new promoter made into pSB1A2 that contains [http://partsregistry.org/Part:BBa_J119044 BBa_J119044]'''<br>  <br>
 
'''Protocol to insert new promoter made into pSB1A2 that contains [http://partsregistry.org/Part:BBa_J119044 BBa_J119044]'''<br>  <br>
  
'''From Oligos'''
+
'''From Boiled and Cooled Oligos'''
 
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> <br>
 
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> <br>
 +
If you don't have time to ethanol precipitate and quantify your DNA, you can use this shortcut:
 +
1 µL cooled oligos.
 +
99 µL water to make a 100X dilution.
 +
 +
Use 1 µL of the 100X diluted oligos and add to the 9 µL described below.
  
 
OR
 
OR
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1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube''). If you are using [http://partsregistry.org/Part:BBa_J119022 BBa_J119022], use 50 ng in 1 µL volume. <br>  
 
1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube''). If you are using [http://partsregistry.org/Part:BBa_J119022 BBa_J119022], use 50 ng in 1 µL volume. <br>  
 
9 µL ligations mixture provided by your instructor. <br>  
 
9 µL ligations mixture provided by your instructor. <br>  
use the promoter DNA for the ligation tube but use only water for the plasmid only tube. <br> <br>  
+
Use the promoter DNA for the ligation tube but use only water for the plasmid only tube. <br> <br>  
  
 
Ligation mixture contains:<br>
 
Ligation mixture contains:<br>
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Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br>  
 
Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br>  
 
Program it for the following cylces: <br>  
 
Program it for the following cylces: <br>  
30 cycles of 37C for 1 minute/16C for 1 minute <br>  
+
20 cycles of 37C for 1 minute/16C for 1 minute <br>  
 
1 cycle of 37C for 15 minutes <br>
 
1 cycle of 37C for 15 minutes <br>
 
22C holding temperature <br>  
 
22C holding temperature <br>  
 
This DNA ligation is ready for transformation. <br> <br>  
 
This DNA ligation is ready for transformation. <br> <br>  
 +
 +
You can increase the cycles to 30 if you want to increase yield. However, we have gotten good success with only 5 cycles.
  
 
'''Transformations''' <br>  
 
'''Transformations''' <br>  
 
You want to do 3 transformations:<br>
 
You want to do 3 transformations:<br>
a positive control<br>
+
# a positive control that contains known [http://partsregistry.org/Part:BBa_K315008 promoter + RBS + RFP] to be used for comparison RFP expression. <br>
your experimental ligation<br>
+
# your experimental ligation<br>
and your negative control ligation<br><br>
+
# your negative control ligation<br><br>
 
Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. <br>  
 
Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. <br>  
 
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>
 
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>
  
Take one tube of 100 µL competent cells and put 30 µL of 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>  
+
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>  
  
  
This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by students in Biology 111 and Romina Clemente.
+
This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by Annie Wacker.

Revision as of 16:20, 28 July 2012

Protocol to insert new promoter made into pSB1A2 that contains BBa_J119044

From Boiled and Cooled Oligos Go to the Loligator web page to determine how to prepare your oligos to add to the solution described below. br>
If you don't have time to ethanol precipitate and quantify your DNA, you can use this shortcut: 1 µL cooled oligos. 99 µL water to make a 100X dilution.

Use 1 µL of the 100X diluted oligos and add to the 9 µL described below.

OR

From Promoter already cloned in a Plasmid
You can start with BBa_J119022 which contains a promoter that is flanked by BsaI sites. Use 50 ng of this plasmid for your "insert" DNA to be added to the ligation mixture listed below.

Get two new, small microfuge tubes designed to fit into the PCR machine.
One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes.


1 µL diluted oligos cooled overnight (use water instead for "plasmid only" tube). If you are using BBa_J119022, use 50 ng in 1 µL volume.
9 µL ligations mixture provided by your instructor.
Use the promoter DNA for the ligation tube but use only water for the plasmid only tube.

Ligation mixture contains:
1 µL (50 ng) plasmid containing part J119044
5 µL dH2O
1 µL 10X Promega Ligase Buffer
1 µL 500 mM KOAc
0.5 µL Bsa I high fidelity (HF) restriction enzyme
0.5 µL T4 DNA Ligase
9 µL final volume

Turn on the PCR machine. Put both of your tubes into the machine.
Program it for the following cylces:
20 cycles of 37C for 1 minute/16C for 1 minute
1 cycle of 37C for 15 minutes
22C holding temperature
This DNA ligation is ready for transformation.

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

Transformations
You want to do 3 transformations:

  1. a positive control that contains known promoter + RBS + RFP to be used for comparison RFP expression.
  2. your experimental ligation
  3. your negative control ligation

Use all 10 µL of the ligations for a transformation.
You will want to perform a transformation positive control using K315008 which contains pLacI+RBS+RFP in plasmid pSB1A2.

Use between 20 - 50 µL of Zippy competent cells into each of three 1.5 mL tubes labeled appropriately for each of the three transformations listed above. Plate all three transformations on LB amp plates.


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