Golden Gate Assembly for Bio113 - Revision history

Macampbell at 14:02, 5 February 2020

2020-02-05T14:02:10Z

2018-05-17T20:30:33Z

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@@ Line 20: / Line 20: @@
 Program it for the following cylces: <br>
 * 20 cycles of 37C for 1.5 minute and 16C for 1.5 minute <br>
 * 22C holding temperature <br>
 * heated lid on

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 * 6 µL dH<sub>2</sub>O <br>
 * 1 µL 10X Promega Ligase Buffer<br>
-* 0.5 µL Bsa I high fidelity (HF) restriction enzyme<br>
+* 0.5 µL Bsa I high fidelity (HFv2) restriction enzyme<br>
 * <u>0.5 µL T4 DNA ligase from Promega</u><br>
 * 9 µL final volume <br> <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>
-* 20 cycles of 37C for 1.5 minute/16C for 1.5 minute <br>
+* 20 cycles of 37C for 1.5 minute and 16C for 1.5 minute <br>
 * 22C holding temperature <br>
 * heated lid on

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 * 20 cycles of 37C for 1.5 minute/16C for 1.5 minute <br>
 * 22C holding temperature <br>
 This DNA ligation is ready for transformation. <br> <br>

@@ Line 19: / Line 19: @@
 Turn on the thermocycler. Put '''both''' of your tubes into the machine. <br>
 Program it for the following cylces: <br>
-* 20 cycles of 37C for 1 minute/16C for 1 minute <br>
+* 20 cycles of 37C for 1.5 minute/16C for 1.5 minute <br>
 * 22C holding temperature <br>
 This DNA ligation is ready for transformation. <br> <br>

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 '''GGA mixture contains:'''<br>
-* 1 µL (40 nM) plasmid containing your receiving plasmid (pClone, or rClone, or actClone, or repClone)<br>
+* 1 µL (40 nM) plasmid containing your receiving plasmid (pClone)<br>
 * 6 µL dH<sub>2</sub>O <br>
 * 1 µL 10X Promega Ligase Buffer<br>

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 * 9 µL final volume <br> <br>
-Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br>
+Turn on the thermocycler. Put '''both''' of your tubes into the machine. <br>
 Program it for the following cylces: <br>
 * 20 cycles of 37C for 1 minute/16C for 1 minute <br>
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 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.

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 ==Starting with Boiled then Cooled Oligos==
-# You should have already used the [http://gcat.davidson.edu/iGem10/index.html Oligator] to design your oligos for your dsDNA promoter. <br>
+# You should have already used the [http://gcat.davidson.edu/iGem10/index.html Oligator] to design your oligos for your dsDNA control element. <br>
 # You should have already calculated how to dilute your boiled and cooled oligos from 5 µM to 40 nM. <br>
 ==Protocol Details for GGA==
-# You will be given two tubes. One tube will be labeled "P" for promoter ligation, the other "-" for the negative control of 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.
+# 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.
-# Add 1 µL '''diluted''' oligos cooled overnight to the tube labeled '''P''' for "GGA promoter".
+# Add 1 µL '''diluted''' oligos cooled overnight to the tube labeled '''X''' for the experimental DNA.
-# Add 1 µL '''water''' to the tube labeled '''-''' for "GGA negative control"
+# Add 1 µL '''water''' to the tube labeled '''N''' for the negative control"
 '''GGA mixture contains:'''<br>