Thursday, January 15, 2015

1 14 2015 DNA Sonication and Quality Checks

I sonicated the DNA on Friday on samples that had a visibly higher quality from the original DNA isolation. The sample were as follow:

Plate NumberD4B4G4H3E5D6G5G6H9D9A9G10
Volume for 500 ng6.546.876.336.89.2810.186.0110.778.
Water to Add43.4643.1343.6743.240.7239.8243.9939.2341.140.340.9740.51
Final Volume (ul)505050505050505050505050
Concentration (ng/ul)101010101010101010101010

Once elutided into 50 ul of nanopure water with 500 ng of DNA the samples were sonicated using the the sonication machine in the Seeb lab (make/model?) with the following protocol. 

In 4C water
3 cycles
60 second on
30 seconds off

After that I waited until yesterday to do a quality check due to time constraints

I used the following protocol to check the gel.

Making a 1.3% agarose gel:
  1. 150 ml 1X TAE in a flask
  2. 2 g agarose mixed into TAE
  3. 5 ul SybrSafe mixed into Agarose
  4. Pour into mold with 15 well comb
  5. Allow gel to stiffen

Loading the Gel:
  1. 10 ul O’GeneRuler 100 bp Ladder in First and Last Well
  2. 20 ul sample mix with 5 ul 1X loading dye
  3. Load 25 ul dye/sample in each well

Running gel:
  1. 120 v for 2 hours
  2. Visualize with Dark reader with orange filter.
  3. Image for notebook

The samples were laid out on the gel in the following manner:
Ladder NF05 NF07 NF12 NF15 HL24 HL26 HL28 HL31 SN01 SN04 SN08 SN15 Ladder

After talking with Carita, Ryan, and Garret today I decided not to move forward with the bisulfite treatment and sequencing because of the poor quality of DNA.

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