Comet in practice 

نوع الشريحه اللي استعملتيها ايه . جربنا نوعين من الشرايح وواحده فقط اللي ما يثبت عليها الجل

For Bacteria, I think, you will need to do dilution for the number of cells because it is smaller in size. I think it would be the same buffers

Isolation and purification of nuclei from mouse brain buffers STM solution (homogenization buffer ) (250mM sucrose + 50 mM Tris- Base + 5mM MgCl2) 2.1 gm sucrose + 0.2 gm tris base Adjust pH at 7.4 by using conc.HCl or NaOH Add 0.01gm MgCl2 then complete the volume to 25 ml Sucrose cushion (1.8M sucrose + 10mM Tris base +3mM MgCl2) 15.4gm sucrose + 30.3 mg tris base Adjust pH at 8 by using conc.HCl or NaOH Add 7.1mg MgCl2 then complete the volume to 25 ml S-buffer (25mM KH2PO4 +1 mM MgCl2+ 1 M sorbitol ) 85 mg KH2PO4 + 2.4 gm MgCl2 + 4.5 gm sorbitol Comet assay buffers Lysing solution (2.5 M NaCl+100 mM EDTA+10 mM Tris-Base) 1- 146.1 gm NaCl + 37.2 gm EDTA+1.2 gm Tris-Base 2- Dissolve in about 700 mL dH2O and begin stirring the mixture Adjust pH at 10 by using conc.HCl or NaOH then complete to 1000 mL Before 30 mins of slide addition, add 89 mL lysing buffer+1 mL of fresh 1%fresh triton X-100 and 10 mL of 10%DMSO Electrophoresis buffer (10N NaOH+200 mM EDTA) Stock solutions stable for 2 weeks 1: 10 N NaOH 200 gm NaOH/500 mL dH2O 2: 200 mM EDTA 14.89 gm EDTA/200 mL dH2O, pH 10 For 1X buffer (prepared fresh before each electrophoresis run). Per liter, 30 mL NaOH + 5 mL EDTA, complete to 1000 mL. pH of the solution must be greater than 13. Neutralization buffer (0.4 M Tris-Base) 48.5 gm Tris-Base 800 mL d H2O Adjust pH 7.5 with concentrated HCl, complete volume to 1000 mL then stored at room temperature. Staining solution (10X) (200 µg/mL) 10 mg Ethidium Bromide/50 mL dH2O Store at room temperature For 1X solution (20 µg/mL), mix 1 mL from 10X stock with 9 mL dH2O Normal melting point agarose (1%) 500 mg dissolved in 50 mL dH2O Low melting point agarose (0.5%) 250 mg dissolved in 50 mL PBS.

Procedures of nuclei mouse brain isolation:  Freshly collected tissues were subjected to glass homogenizer with 750ul of homogenization buffer  Homogenized each sample 21 times with loose pestle  750ul of sucrose cushion was added to the bottom of the centrifuge tube  Homogenates (750ul) were layered on the sucrose cushion  Third layer was 750ul STM buffer  The tubes were centrifuged at 5000 rcf at 40C for 30mins  The supernatant was carefully removed by aspiration  Nuclei re-suspended in STM buffer   Nuclei were re-suspended in 20ul of s-buffer Comet assay procedure Preparation of frosted microscope slides  The slides were dipped into methanol and burned over a blue flame to remove the machine oil and dust  Slides dipped into a warmed normal melting agarose (NMA) and gently removed  Nuclei solution mixed with 740ul of 0.5% LMPA  The nuclei were spread on frosted microscope slides pre-coated with a layer of 1% normal melting agarose.  Third layer of 0.5% LMPA spreaded on frosted microscope slides  After gelling, the slides were treated with lysing buffer for one hour at 4ºC.  Slides were then placed in the electrophoresis solution for 20 min to allow DNA unwinding.  Electrophoresis was carried out at 24 V and 300 mA for 30 min. The slides were then neutralized with neutralization buffer, for 5 min/3 times  Drained slides were kept in cold 100% methanol for 20mins  Stained with ethidium bromide (200 μg/mL).  All these steps were performed in the dark to prevent additional DNA damage.  The pictures were taken by Olympus BX43

In order to do it from whole blood, you will need to lyse the red blood cells and wash then proceed with the cells as in the video Only the tissue samples require STM buffer to get a clean nuclei

وعليكم السلام fkaneer@mans.edu.eg +201551919531