Seahorse DNA Extraction

Procedure

• Solution preparation• Sample preparation• Methods to extract DNA:

Phenol: ChloroformSalting out: with Chloroform and without

Chloroform

Result

First result in 29/9/2010. Eight wells were loaded with DNA solution, four wells showed the presence of DNAs (phenol: chloroform method). Gel loading in 40 mins, 150V, SB (pH= 8.0) buffer, 2% agarose gel.

Result in 8/10/2010. Eight wells were loaded with DNA solution, only the 3rd well showed the presence of DNAs (phenol: chloroform method). Gel loading in 20 mins, 120V, SB (pH= 8.0) buffer, 1% agarose gel.

Gel loading in 11/10/ 2010

The first line: DNA prepared by phenol: chloroform method (9 samples), the second line: the first 4 wells, samples made by salting out with Chloroform, the rest was by salting out without Chloroform. The left- hand side picture: gel running in 15 mins, 100V, the right- hand side picture: 15 more mins.

Gel running in 40 mins, 100V. Agarose gel 1.5%, SB buffer (pH= 8)

Gel loading in 16/10/ 2010

Summary

• Cells/ Tissues from muscle and fin. From muscle as well as fin, the quantity of samples ought to be small to avoid hurting or even killing a live specimen. Muscle should be taken in the tail, especially around the last tail ring.

• Muscle taking by aseptic methods and fin preparation by fin- clipping.

• Individual Protocol:Taking a small amount of muscle (10<m<20

(mg)) or fin (the size: 1x2 or 2x2mm), chop it into very small pieces.

Adding TNES buffer and Protease (or Proteinase K) to lysis the cells. The volume’s 300µl of TNES and 20µl of protease (10µl of Proteinase K).

Incubating the sample(s) in 56oC, about 18 hours to overnight.

Phenol: Chloroform method Salting out without Chloroform

Salting out with Chloroform

Centrifugation done in 30 mins, max. speed 12,000 to 14,000 rpm, 25oC ( for salting out method with Chloroform, 300 to 400µl of Chloroform and (the total volume of solution/ 3.8) NaCl 6M will be put in the solution).

Remove supernatant into a new tube. Then putting the volume of Phenol: Chloroform: Isoamylpropanol is similarly equal to the volume of the supernatant.

Adding 1/10 volume of NaAc 3M into the supernatant after taking out it into new tube(s). Then placing it into -20oC freezer in 20 mins.

Taking out the upper layer of solution the volume can reach to the max. volume into new tube(s).

Doing centrifugation in 20 mins, 12,000- 14,000 rpm, 4oC Adding 2 volumes of Isopropanol or cold absolute Ethanol, then shake it well.

Taking the possible max. volume of the upper phase in the solution into new tube(s).

Discard the pellets at the bottom.

Adding 2 volumes of Isopropanol or cold absolute Ethanol, then shake it well.

The 3 methods recommended to place all tubes in the -20oC freezer overnight to gain more DNA pellets.

• After incubating DNA prepared by 3 methods in -20oC freezer, take them out, carrying out centrifugation in 20 mins, 12,000- 14,000rpm, 4o C.

• Discard supernatant, then wash the pellets by EtOH 96o , centrifuge the tube(s) in 5 mins, max. speed, 4oC. Wash the pellets again with EtOH 70o , centrifuge the tube(s) in 5 mins, max. speed, 4oC.

• Let all the tubes dry in room temp. at least 5 hours or

overnight.

Discussion

The DNA gained above is not as good as expected. All the bands showed not clear and most of them were smear. The DNA could be broken somewhere in some step of doing the procedure.