Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2012/08/29: Difference between revisions
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==Digestion== | ==Digestion== | ||
* We usually use ECO-R1 or PST-1 to cut (refer to Dr. Hayes Biobrick cloning 12/16/09) | * We usually use ECO-R1 or PST-1 to cut (refer to Dr. Hayes Biobrick cloning 12/16/09) | ||
* Part: BBa_J176122, Vector: MV2, Transfection Plasmid: KAH126 | * Part: [http://partsregistry.org/wiki/index.php?title=Part:BBa_J176122 BBa_J176122, Vector: MV2], Transfection Plasmid: [http://partsregistry.org/wiki/index.php?title=Part:BBa_J176062 KAH126] | ||
<br> | <br> | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table --> | ||
|-valign="top" | |-valign="top" | ||
| <u>Reagent</u> || <u>Volume</u> || <u> Master Mix </u> | | <u>Reagent</u> || <u>Volume</u> || <u> Master Mix </u> | ||
| rowspan="7" | <u>Expected:</u><br> | | rowspan="7" | <u>Expected:</u><br> KAH126 = 1880bps <br> MV2 = 4529bps<br> | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:CA_Gel.jpeg|300px|E/P digests 8/29/12]]<br>15 μL/lane; 1% agarose | ||
| rowspan ="7" | [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]<br>Gene Ruler | |||
|- | |- | ||
| DNA(plasmid) || 3.0 μL || - | | DNA(plasmid) || 3.0 μL || - | ||
Line 25: | Line 26: | ||
|- | |- | ||
| dH<sub>2</sub>O || 8.5 || 25.5 | | dH<sub>2</sub>O || 8.5 || 25.5 | ||
|} | |} | ||
*Use two 0.5mL tubes for single tubes and a 1.5ML tube for the MM | |||
# Aliquot 12uL to each 0.5mL tube | |||
# Add 3 uL of DNA to each | |||
# Incubate @ 37C/>10min | |||
<br> | |||
How to make a gel: | |||
# 60mL of TAE and 0.6g of agarose, swirl | |||
# 40s in the microwave, swirl | |||
# 40s in the microwave, swirl | |||
# 6mL of Ethidium Bromide | |||
*Note: Wash flask immediate | |||
<br> | |||
Moving onto the box | |||
*If there is TAE buffer in the electrophoresis, put box in a casting tray | |||
*Use B14 teeth (thinner side, thicker side is for dilute DNA). We had 2 rows. | |||
*Check for bubbles in wells and push them out of the wells if needed | |||
<br> | |||
Gel Electrophoresis Procedure | |||
*It takes 15-20 min for the gel to solidify | |||
*At 70V is takes about 1hr for the gel to run | |||
*Gel is done solidifying when it's translucent | |||
#Grab comb out from both sides | |||
#Put in runner | |||
#Add 10uL of Ethidium Brodmide to TAE at positive end (the end facing you) | |||
#1KB+ loading dye (blue) > 10uL of ladder > 15uL of sample | |||
#Pour off extra liquid and set gel on a paper towel | |||
#Slide gel off onto UV light | |||
#Put gel in waste box | |||
#SIZES! Compare to expected (MV2, HPCD...) > 2 bands | |||
<br> | |||
Analyzing the Gel | |||
*As a sanity check, prep 1 is more concentrated (as shown in the plate reading) and prep 2 is less bright because it is less concentrated | |||
*Our digest was done correctly. Checking with the ladder, the bands are lining up around the expected distances. (Check the 1500 and 4000 band lines on the ladder) | |||
*We see two bands because the plasmid was cut twice | |||
Revision as of 17:45, 5 September 2012
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Digestion
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