Purdue/16 October 2008

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Latest revision as of 15:05, 16 October 2008

Performed another ligation (for protocol, see Sept. 25). It turns out that the competent cells we used before are lacZ+. :(

Therefore, both UV-exposed and non-exposed bacteria were turning blue because they were on X-gal plates.

We were able to try a different method of transformation: Dr. Clase provided us with some Bio-Rad Hb101 cells (which naturally take up plasmids). We are following the Bio-Rad protocol for transformation:

Put 250uL transformation solution (CaCl2) into an eppendorf tube
Put tube on ice
Add colony from starter plate of Hb101 cells to tube
Incubate on ice 10 min.
Heat shock at 42C for 50 seconds
QUICKLY move back to ice and incubate for 2 minutes
Remove from ice, add 250uL LB broth, incubate at RT for 10 min.
Tap to mix, plate 100uL on amp plate

Edited by Janie Stine

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 [[Team:Purdue/Notebook | Click Here to return to the notebook.]]
+Performed another ligation (for protocol, see Sept. 25).
+It turns out that the competent cells we used before are lacZ+.  :(
+Therefore, both UV-exposed and non-exposed bacteria were turning blue because they were on X-gal plates.
+We were able to try a different method of transformation:  Dr. Clase provided us with some Bio-Rad Hb101 cells (which naturally take up plasmids).  We are following the Bio-Rad protocol for transformation:
+# Put 250uL transformation solution (CaCl2) into an eppendorf tube
+# Put tube on ice
+# Add colony from starter plate of Hb101 cells to tube
+# Incubate on ice 10 min.
+# Heat shock at 42C for 50 seconds
+# QUICKLY move back to ice and incubate for 2 minutes
+# Remove from ice, add 250uL LB broth, incubate at RT for 10 min.
+# Tap to mix, plate 100uL on amp plate
+'''Edited by Janie Stine'''