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

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(Digestion)
Current revision (19:45, 5 September 2012) (view source)
(Digestion)
 
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{| 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> Expected </u>
+
| <u>Reagent</u> || <u>Volume</u> || <u> Master Mix </u>  
| rowspan="7" | <u>Expected:</u><br> KAH126 = 1880bps <br> MV2 = 4529bps<br>
| rowspan="7" | <u>Expected:</u><br> KAH126 = 1880bps <br> MV2 = 4529bps<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

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Digestion


Reagent Volume Master Mix Expected:
KAH126 = 1880bps
MV2 = 4529bps
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
  • 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


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


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