Difference between revisions of "Golden Gate Assembly protocol"
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| − | '''Protocol to insert new promoter made from oligos into pSB1A2 with insert [http://partsregistry.org/Part:BBa_J100028 BBa_J100028]'''<br> | + | '''Protocol to insert new promoter made from oligos into pSB1A2 with insert [http://partsregistry.org/Part:BBa_J100028 BBa_J100028]'''<br> <br> |
| − | 1 µL | + | |
| − | 1 µL (10 ng) pSB1A2+J100028<br> | + | First, you need to dilute your assembled oligos. To do this, dilute them 200 fold. <br> |
| − | 5 µL | + | The easiest way to do this is to remove 2 µL from the 20 µL overnight mixture and place this 2 µL into a new, small microfuge tube. <br> |
| + | Now add 398 µL water to the 2 µL of cooled, annealed ologs. Mix this by shaking the tube (lid closed of course). <br> <br> | ||
| + | |||
| + | Get '''two''' new, small microfuge tubes designed to fit into the PCR machine. <br> | ||
| + | One tube will be labeled "ligation", the other "plasmid only".<br> <br> <br> | ||
| + | 1 µL diluted oligos cooled overnight (''leave this out for plasmid only'')<br> | ||
| + | 1 µL (10-20 ng) pSB1A2+J100028<br> | ||
| + | 5 µL dH<sub>2</sub>O (''6 µL for plasmid only'')<br> | ||
1 µL 10X Promega Ligase Buffer<br> | 1 µL 10X Promega Ligase Buffer<br> | ||
1 µL 500 mM NaCl<br> | 1 µL 500 mM NaCl<br> | ||
| − | 0.5 µL Bsa I restriction enzyme<br> | + | 0.5 µL Bsa I high fidelity (HF) restriction enzyme<br> |
<u>0.5 µL T4 DNA Ligase</u><br> | <u>0.5 µL T4 DNA Ligase</u><br> | ||
10 µL final volume (be sure to perform a negative control ligation) <br> <br> | 10 µL final volume (be sure to perform a negative control ligation) <br> <br> | ||
| − | Turn on the PCR 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> | ||
40 cycles of 37C for 1 minutes/16C for 1 minutes <br> | 40 cycles of 37C for 1 minutes/16C for 1 minutes <br> | ||
| Line 19: | Line 26: | ||
Use all 10 µL of the ligations for a transformation. <br> | Use all 10 µL of the ligations for a transformation. <br> | ||
You will want to do a negative control where you leave out the assembled oligos and add one more microliter of water. <br> | You will want to do a negative control where you leave out the assembled oligos and add one more microliter of water. <br> | ||
| − | You will also want to perform a positive control of [http://partsregistry.org/Part:BBa_K315008 K315008] which contains pLacI+RBS+RFP in plasmid pSB1A2. <br> | + | You will also want to perform a positive control of [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 use 30 µL of cells 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> | Take one tube of 100 µL competent cells and use 30 µL of cells 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> | ||
Revision as of 12:32, 31 August 2011
Protocol to insert new promoter made from oligos into pSB1A2 with insert BBa_J100028
First, you need to dilute your assembled oligos. To do this, dilute them 200 fold.
The easiest way to do this is to remove 2 µL from the 20 µL overnight mixture and place this 2 µL into a new, small microfuge tube.
Now add 398 µL water to the 2 µL of cooled, annealed ologs. Mix this by shaking the tube (lid closed of course).
Get two new, small microfuge tubes designed to fit into the PCR machine.
One tube will be labeled "ligation", the other "plasmid only".
1 µL diluted oligos cooled overnight (leave this out for plasmid only)
1 µL (10-20 ng) pSB1A2+J100028
5 µL dH2O (6 µL for plasmid only)
1 µL 10X Promega Ligase Buffer
1 µL 500 mM NaCl
0.5 µL Bsa I high fidelity (HF) restriction enzyme
0.5 µL T4 DNA Ligase
10 µL final volume (be sure to perform a negative control ligation)
Turn on the PCR machine. Put both of your tubes into the machine.
Program it for the following cylces:
40 cycles of 37C for 1 minutes/16C for 1 minutes
80C for 15 minutes
22C holding temperature
This DNA ligation is ready for transformation.
Transformations
Use all 10 µL of the ligations for a transformation.
You will want to do a negative control where you leave out the assembled oligos and add one more microliter of water.
You will also want to perform a positive control of K315008 which contains pLacI+RBS+RFP in plasmid pSB1A2.
Take one tube of 100 µL competent cells and use 30 µL of cells 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 students in Biology 111 and Romina Clemente.
