Comet in practice
Вставка
- Опубліковано 9 вер 2024
- Demonstrated by: M.Sc. Miram Altouhamy
Recorded by: M.Sc. Abeer Alsaudi
Edited by: Researcher Youssef Darwish
Supervision: Dr. Fardous ElSenduny
The video is a demonstration for the following article:
Dhawan, Alok, et al. "Protocol for the single cell gel electrophoresis/comet assay for rapid genotoxicity assessment." Sigma 1077.1 (2003): 1-10.
جربت طريقتك ولكن الجل ينفك من عالشريحة بعد عملية الرحلان الكهربي فهل الخلل في تحضير الجل وكذلك محلول التحميل او الالكالاين بفر عمل رغاوي بعد تشغيل الجهاز مع انه نفس خطوات التحضير اللي منزلتيه ولقيت نفس الطريقة بالضبط في jove
نوع الشريحه اللي استعملتيها ايه . جربنا نوعين من الشرايح وواحده فقط اللي ما يثبت عليها الجل
ممكن حضرتك تشرحي ازاى اجهز عينه الخلايا وليكن من الدم او النسيج...............اكون شاكرة فضلك
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
Can I do the comet assay using bacterial cells? I am having problem finding correct lysis solutions.
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
السلام عليكم دكتور ممكن ايميل حضرتك للتواصل مع حضرتك
وعليكم السلام
fkaneer@mans.edu.eg
+201551919531