"
Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period3/Dailylog
From 2013.igem.org
| Line 55: | Line 55: | ||
- Made new LB and x6 top agar. | - Made new LB and x6 top agar. | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<br> | <br> | ||
| Line 66: | Line 60: | ||
<font size="4"> '''6/3/13''' </font> | <font size="4"> '''6/3/13''' </font> | ||
| - | - | + | -KK KP Our PCR product was consistent with every other attempt to amplify CRO: it failed. Consistency is only a virtue if you're not a screw-up :) Anyway, our gel showed a faint band at the 300 base pair level, but too faint to be considered successful. We considered a list of reasons why our PCR reaction is failing and discussed them with Dr. Grose: |
| - | + | We boiled our template DNA for 10 minutes, but Dr. Grose recommends 5 minutes | |
| - | - | + | We added 1 microliter of template; Dr. Grose likes to add 2 microliters. |
| + | We did not use a master mix to combine our reagents with our template DNA. We added each reagent individually to each tube. Because we are pipetting such small volumes, there is a lot of room for error this way. | ||
| + | The Phusion program we used was set to an annealing temperature that was 1 degree too low for Phusion. | ||
| + | We did the PCR reaction one more time with Dr. Grose. Tomorrow we expect to see an enormous, fat, band! | ||
<br> | <br> | ||
Revision as of 19:15, 7 June 2013
| Cholera Detection May - June Notebook: May 27 - June 9 Daily Log
| ||
|
|
5/28/13 - Started three 8mL E coli BL21 liquid culture at around 4pm.
5/29/13 -KP KK Today we plated TT9907 and TT9901. We plated them on LB arabinose/amp/x-gal plates. We plated different concentrations of TT9907 and TT9901 on each of the plates. We added 50, 100, and 300 microliters onto each plate. We will check the plates tomorrow hopefully to find plagues showing that arabinose triggered lysis of our lambda. We unfortunately didn't get any growth of our TT23821, so we streaked another plate from our original stab sample and we will try to re-electroporate TT23821 on Friday.
5/30/13 - Plates from yesterday are taken out of incubation at around 4:00pm
5/31/13 - Discussed results for 5.20 Mutagen Concentration Experiment -> will need to try this one more time with minor adjustment to experimental design: plate -4 dilution using x6 and x8 top agar. - Discussed plans for next week. - Made new LB and x6 top agar.
6/3/13 -KK KP Our PCR product was consistent with every other attempt to amplify CRO: it failed. Consistency is only a virtue if you're not a screw-up :) Anyway, our gel showed a faint band at the 300 base pair level, but too faint to be considered successful. We considered a list of reasons why our PCR reaction is failing and discussed them with Dr. Grose: We boiled our template DNA for 10 minutes, but Dr. Grose recommends 5 minutes We added 1 microliter of template; Dr. Grose likes to add 2 microliters. We did not use a master mix to combine our reagents with our template DNA. We added each reagent individually to each tube. Because we are pipetting such small volumes, there is a lot of room for error this way. The Phusion program we used was set to an annealing temperature that was 1 degree too low for Phusion. We did the PCR reaction one more time with Dr. Grose. Tomorrow we expect to see an enormous, fat, band!
6/5/13 Regarding our PCR attempt yesterday, we did see a thick fat band ... at the bottom of the gel! Once again our PCR failed. This has led us to propose several reasons why we can't PCR amplify CRO: 1) the Lambda prophage isn't actually in TT 9901 or TT9907, or 2) our primers aren’t functioning to amplify CRO. We streaked a few plates of TT9901 and TT9907 to subject to UV light. If we see plaques, then we will know Lambda has been induced. Meanwhile Dr. Grose is going to check that our primers are the correct ones.
| |
