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 Number	D4	B4	G4	H3	E5	D6	G5	G6	H9	D9	A9	G10
Sample	HL28	HL26	HL31	HL24	NF05	NF12	NF07	NF15	SN08	SN04	SN01	SN15
Volume for 500 ng	6.54	6.87	6.33	6.8	9.28	10.18	6.01	10.77	8.9	9.7	9.03	9.49
Water to Add	43.46	43.13	43.67	43.2	40.72	39.82	43.99	39.23	41.1	40.3	40.97	40.51
Final Volume (ul)	50	50	50	50	50	50	50	50	50	50	50	50
Concentration (ng/ul)	10	10	10	10	10	10	10	10	10	10	10	10

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

cycle=

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.