Minnesota/1 August 2008

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Latest revision as of 20:11, 4 August 2008

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1. Ran the terminator digest out on a gel: After which we were unable to see a band the appropriate length, so we simply cut out the gel where we expected the band to be (terminator is very small - so hard to see). Gel purified - then used this in our next ligation. When spec'ed the purified gel terminator product, there were 2.5ngram/uL of DNA concentration.

2. Currently have two parts that are base vector-promoter-gene, and one that is base vector-promoter: We are currently ligating the terminator to the two parts that have vector-promoter-gene.

The base vector-promoter-gene constructs are: TetR promoter:Lambda cI and Tet/p22 promoter:RFP.

The base vector-promoter construct is: Lambda cI/Lac promoter

3. Digest Rxn: Digested Terminators, Lac/LAMBDAcI, and GFP. Follow the table below:

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2	Total
Term 2	5.0uL	0.5uL	39.5uL	3.0uL	1.0uL, Xba1	1.0uL, Pst1	50.0uL
Term 4	5.0uL	0.5uL	28.5uL	14.0uL	1.0uL, Xba1	1.0uL, Pst1	50.0uL
Lac/LAMBDA A	5.0uL	0.5uL	8.0uL	34.5uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
Lac/LAMBDA B	5.0uL	0.5uL	16.0uL	26.5uL	1.0uL, Spe1	1.0uL, Pst1	50.0uL
GFP	5.0uL	0.5uL	40.5uL	2.0uL	1.0uL, Xba1	1.0uL, Pst1	50.0uL

@@ Line 15: / Line 15: @@
 |-
 |The base vector-promoter construct is: Lambda cI/Lac promoter
+|-
+|'''3. Digest Rxn:''' Digested Terminators, Lac/LAMBDAcI, and GFP. Follow the table below:
+{|border="1" align="left"
+|-
+!| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2 !! Total
+|-
+| Term 2 || 5.0uL || 0.5uL || 39.5uL || 3.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
+|-
+| Term 4 || 5.0uL || 0.5uL || 28.5uL || 14.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
+|-
+| Lac/LAMBDA A || 5.0uL || 0.5uL || 8.0uL || 34.5uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| Lac/LAMBDA B || 5.0uL || 0.5uL || 16.0uL || 26.5uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
+|-
+| GFP || 5.0uL || 0.5uL || 40.5uL || 2.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
+|}