Corum:DNA Hybridization: Difference between revisions

Corum:DNA Hybridization (view source)

130 bytes removed , 24 July 2012

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 # Cool on bench 5 min.
 # Place weighted holder with sealed oligonucleotides into the beaker.
-# Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into dsDNA linker at 1mM final concentration.
+# Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into as single dsDNA linker with sticky ends at 1mM final concentration.
-# Dilute the 1mM linker 1/10 (~10 μL 1mM linker into ~ 90 μL H<sub>2</sub>O, may need to be adjusted if some volume is lost and, for example only 9 μL 1mM linker is recovered). Mix and spin.
+# Perform two 1/100 dilutions down to 100 nM (label all tubes clearly):
-# Continue 1/10 dilutions down to 100 nM (label all tubes clearly):
+#* 2 μL 1mM linker + 198 μL H<sub>2</sub>O = 200 μL 10μM linker
-#* 20 μL 100μM linker + 180 μL H<sub>2</sub>O = 200 μL 10μM linker
+#* 2 μL 10μM linker + 198 μL H<sub>2</sub>O = 200 μL 100nM linker
-#* 20 μL 10μM linker + 180 μL H<sub>2</sub>O = 200 μL 1μM linker
+# Store 1mM, 10μM, and 100nM stocks at -40 °C in latest "Primer" box. Keep an aliquot of the 100nM linker in your own box for use in constructions.
-#* 20 μL 1μM linker + 180 μL H<sub>2</sub>O = 200 μL 100nM linker
-# Store at -40 °C in latest "Primer" box.
 ==Notes==