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August 11th, 2009
Amplification of the promoter and RBS using plasmidic DNA from Q04121.
Digestion Mix PlasmidicDNA 4.0 μl (= 1 μg DNA) x 20 = 80 μl BSA 0.5 μl x 20 = 10 μl Buffer 2 1.0 μl x 20 = 20 μl Enzyme EcoRI 0.5 μl x 20 = 10 μl H2O 4.0 μl x 20 = 80 μl Cf 10.0 μl 200 μl
Load order:
Well Plasmid 2 2:3 L1 3 2:3 L2’ 4 2:3 L2” 5 15:13 6 15:3 7 4:23 8 4:23 L2
Gel 7:31 pm Well Plasmid Well Plasmid 1 Marker 1 Marker 2 2_3L2#1 2 9.1 3 2:3L2#2 3 9.3 4 2_3L2#3 4 9.4 5 3.3* 5 9.5 6 4_23L1 6 9.6 7 No 4_23L2 7 9.7 8 7.3 8 9.8 9 11.1 9 9.9 10 14.2 10 9.10 11 15.3 11 Marker 12 15_13L2 #1 12 ---------- 13 15_13L2 #2 Digest with EcoRI 14 15_13L2 #3 at 4°C 15 16.2” Digest with EcoRI 16 18.2* 17 19.1* Repeat digestion –DNA 18 22.1* Repeat digestion-DNA 19 24.2” Repeat digestion – DNA 20 Marker Note: it is needed to digest lesser the plasmid 11.1.
Gel 7:31 pm Well Plasmid Well Plasmid 1 Marker 1 Marker 2 2_3L2#1 2 9.1 3 2:3L2#2 3 9.3 4 2_3L2#3 4 9.4 5 3.3* 5 9.5 6 4_23L1 6 9.6 7 No 4_23L2 7 9.7 8 7.3 8 9.8 9 11.1 9 9.9 10 14.2 10 9.10 11 15.3 11 Marker 12 15_13L2 #1 12 ---------- 13 15_13L2 #2 Digest with EcoRI 14 15_13L2 #3 at 4°C 15 16.2” Digest with EcoRI 16 18.2* 17 19.1* Repeat digestion –DNA 18 22.1* Repeat digestion-DNA 19 24.2” Repeat digestion – DNA 20 Marker
Note: it is needed to digest lesser the plasmid 11.1.
Repeat digestions of the plasmids:
which are at -20°C in the Unicel (with 3μl of DNA) and the 9 is at 4°C. Put 3 μl of DNA and 5 μl of water. Digestion Mix x 10.
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