User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22: Difference between revisions
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==Running an Analytical DNA Gel== | ==Running an Analytical DNA Gel== | ||
# Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells). | |||
# When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer. | |||
# Load 5μL of DNA ladder into the 1st well. | |||
# Load 5μL of the [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/21| M8S/M33S/M103S/A71M/L40M PCR product from yesterday]] with 1μL 6x loading buffer into the 3rd well (mix before pipetting into well). | |||
# Load 5μL of the wild-type Asc Hb and pQE-80-L-Kan ligation from [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22| this morning]] with 1μL 6x loading buffer into the 5th well (mix before pipetting into well). | |||
# Load 5μL of the triple mutant (M8S/M33S/M103S) Asc Hb and pQE-80-L-Kan ligation from [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22| this morning]] with 0.5μL 6x loading buffer into the 7th well (mix before pipetting into well). | |||
# Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel | |||
# Place the gel in Ethidium Bromide Stain for about 30 minutes | |||
# Move the gel to TAE buffer to destain for about 20 minutes | |||
# View under a UV light | |||
#* Be careful when working with ethidium bromide and UV light. | |||
==Transformations== | ==Transformations== |
Revision as of 07:42, 22 June 2012
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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
Transformations
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