User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/22: Difference between revisions
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==Continuing Wildtype Hemoglobin Expression== | |||
# Centrifuge cultures grown overnight at 4500rpm at 4°C for 15 minutes. | |||
# Resuspend each pellet in 1mL of LB. | |||
# Add each pellet to 1L of LB with 100μg/mL of ampicillin (four of these). | |||
# Incubate at 37°C at 165rpm until OD<sub>600</sub> = 0.6. | |||
#* Actual OD<sub>600</sub> = 0.64 | |||
# Add 1mL 0.1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) to each flask. | |||
#* Prepared fresh today: 0.12g + 5mL distilled H<sub>2</sub>O, sterile-filter | |||
# Add 37.5mL of hemin solution to each flask. | |||
#* The hemin solution was prepared by [[User:Matt Hartings| Dr. Hartings]] on 6/20/12. It was prepared by combining 160mg hemin with 1.38mL triethanolamine, and diluting the solution to 40mL. The pH of the solution was then adjusted to 7.8, and then water was added to a final volume of 150mL. | |||
# Let cells continue to grow and express for 4 hours at 30°C at 165rpm. | |||
# Spin the cells at 4500rpm at 4°C for 15 minutes. | |||
# Resuspend the cells in sterile-filtered 25mM Tris, 50mM NaCl, pH 8 (35mL total). | |||
# Freeze the cells in liquid nitrogen and store at -20°C. | |||
==Miniprep Growths== | ==Miniprep Growths== | ||
None 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 [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/20|two days ago]]. The plate was just left at room temperature overnight. | None 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 [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/20|two days ago]]. The plate was just left at room temperature overnight. | ||
==DNA ligation with T4 DNA Ligase== | ==DNA ligation with T4 DNA Ligase== | ||
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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. | |||
[[Image:Photo_(18).JPG]] | |||
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== | |||
I need to make sure that there is actually DNA in my mini-preps from [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/19| 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: | |||
# Using a smaller volume quartz cuvette, 95μL of dH<sub>2</sub>O was placed in the spectrophotometer and the baseline was corrected with this. | |||
# 5μL of DNA was added to the 95μL of water. This was pipetted up and down with a pipette to mix. | |||
# The absorbance was taken at 260 and 280nm. | |||
== | ===Results=== | ||
Note: the absorbances are for 20x dilute DNA samples | |||
====Colony 2==== | |||
* A<sub>260</sub> = 0.147 | |||
* A<sub>280</sub> = 0.081 | |||
* Absorbance Ratio = 1.8089 | |||
* 20x Dilute DNA concentration = 7.3350 μg/mL | |||
* Non-dilute DNA concentration = 146.7 μg/mL | |||
====Colony 3==== | |||
* A<sub>260</sub> = 0.181 | |||
* A<sub>280</sub> = 0.104 | |||
* Absorbance Ratio = 1.7404 | |||
* 20x Dilute DNA concentration = 9.0500 μg/mL | |||
* Non-dilute DNA concentration = 181 μg/mL | |||
So it seems there is DNA, which means there are other things that can probably be troubleshooted. | |||
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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. |