User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22: Difference between revisions
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Again, there are no band on the gel. I still may try to transform in case the DNA concentration is low, but usually bands at least show up bright after a successful PCR. | Again, there are no band on the gel. I still may try to transform in case the DNA concentration is low, but usually bands at least show up bright after a successful PCR. | ||
==Quantifying DNA== | ==Quantifying DNA== |
Revision as of 06:26, 25 June 2012
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Continuing Wildtype Hemoglobin Expression
Miniprep GrowthsNone of the cultures started for the minipreps had any growth last night (the "colonies" were very small). This means no minipreps will be done today. Two colonies did however appear on the NovaBlue + M103S transformation plate from two days ago. The plate was just left at room temperature overnight. DNA ligation with T4 DNA LigaseThis will be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert. The procedure is based off of the protocol on the NEB website.
My calculations yesterday led me to a 5:1 volume ratio, but I thought about it again and here is my reasoning for for today for a 3:1 insert to vector molar ratio:
Running an Analytical DNA Gel
Again, there are no band on the gel. I still may try to transform in case the DNA concentration is low, but usually bands at least show up bright after a successful PCR.
Quantifying DNAI need to make sure that there is actually DNA in my mini-preps from Tuesday because my two PCR reactions this week did not seem to work. To do this I will quantify the DNA in the colonies 2 and 3 minipreps of M8S/M33S/M103S/A71M Asc Hb. This procedure was repeated for both minipreps:
ResultsNote: the absorbances are for 20x dilute DNA samples Colony 2
Colony 3
So it seems there is DNA, which means there are other things that can probably be troubleshooted. |