Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2012/08/29

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(Digestion)
(Digestion)
Line 13: Line 13:
|-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>1,2. KAH123 = 8002<br>3,4. KAH124 = 8174<br>5,6. KAH125 = 8246<br>
+
| rowspan="7" | <u>Expected:</u><br>1,2. KAH126 = 1880bps <br>3,4. MV2 = 8174<br>5,6. KAH125 = 8246<br>
-
| rowspan="7" | [[Image:CA_Gel.jpeg|300px|E/P digests 8/29/12]]<br>15 μL/lane; 1% agarose -->
+
| 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 -->
+
| rowspan ="7" | [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]<br>Gene Ruler
|-
|-
| DNA(plasmid) || 3.0 μL || -
| DNA(plasmid) || 3.0 μL || -

Revision as of 15:21, 5 September 2012

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Digestion


Reagent Volume Master Mix Expected:
1,2. KAH126 = 1880bps
3,4. MV2 = 8174
5,6. KAH125 = 8246
E/P digests 8/29/12
15 μL/lane; 1% agarose
Image:KAH_Fermentas_GeneRuler_1kbplus.jpg
Gene Ruler
DNA(plasmid) 3.0 μL -
10X buffer 1.5 4.5
EcoRI 1.0 3.0
PstI 1.0 3.0
dH2O 8.5 25.5
  15 μL --> 37°C/ ~15 min.
  • Use two 0.5mL tubes for single tubes and a 1.5ML tube for the MM
  1. Aliquot 12uL to each 0.5mL tube
  2. Add 3 uL of DNA to each
  3. Incubate @ 37C/>10min


How to make a gel:

  1. 60mL of TAE and 0.6g of agarose, swirl
  2. 40s in the microwave, swirl
  3. 40s in the microwave, swirl
  4. 6mL of Ethidium Bromide
  • Note: Wash flask immediate


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


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
  1. Grab comb out from both sides
  2. Put in runner
  3. Add 10uL of Ethidium Brodmide to TAE at positive end (the end facing you)
  4. 1KB+ loading dye (blue) > 10uL of ladder > 15uL of sample
  5. Pour off extra liquid and set gel on a paper towel
  6. Slide gel off onto UV light
  7. Put gel in waste box
  8. SIZES! Compare to expected (MV2, HPCD...) > 2 bands


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