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Agarose gel electrophoresis of PCR products - Method and Protocol

Agarose gel electrophoresis of PCR products - Method and Protocol - PCR - Polymerase Chain Reaction Forum

Agarose gel electrophoresis of PCR products - Method and Protocol - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 08-01-2006, 12:01 AM
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Post Agarose gel electrophoresis of PCR products - Method and Protocol



Agarose gel electrophoresis of PCR products

protocol by : Robert H. Cruickshank

MATERIALS REQUIRED

PREPARATIONS

PROTOCOL


Table
1xTAE
50xTAE
6x Gel Loading Buffer

NOTES

MATERIALS REQUIRED

* Glass bottles and measuring cylinders up to 1 litre capacity
* Pipettes and tips for handling volumes from 1-100ml
* 242g of Tris Base
* 57.1ml glacial acetic acid
* 100ml 0.5M EDTA pH8.0
* Glycerol
* Bromophenol blue
* Xylene cyanol FF
* Agarose
* Ethidium bromide
* 1kb ladder
* MINI and/or MIDI horizontal electrophoresis tanks with removable gel casting trays, casting gates and combs
* A power supply capable of delivering 150 Volts (or 75 Volts if only using a MINI gel tank)
* High quality (double) distilled water supply
* +4 C refrigerator for quick setting of gels (optional)

PREPARATIONS

Prior to agarose gel electrophoresis it is necessary to make the following preparations:

1. Make up 50xTAE
2. Make up 1xTAE
3. Make up 6x gel loading buffer
4. Place casting gates and combs in gel casting tray on a level surface


PROTOCOL

1. Add the appropriate volume of 1xTAE to a sterile container (see table)
2. Add the appropriate amount of agarose (see table)
3. Heat in a microwave with the lid slightly loosened until boiling (about 2 minutes on full power)
4. Allow to cool to 'hand warm' (50-60 C) e.g. by running under a cold tap
5. Add the appropriate amount of ethidium bromide (see table).

EXERCISE CAUTION WHEN USING ETHIDIUM BROMIDE WHICH IS A KNOWN CARCINOGEN


6. Pour the gel into the gel casting tray
7. Remove any bubbles in the gel
8. Allow the gel at least 40 minutes to set (or less if put in a fridge) ensuring that the gel casting tray is level and undisturbed
9. When the gel has set remove the combs and casting gates and transfer to the gel tank
10. Pour the appropriate amount of 1xTAE into the gel tank so that it covers the gel (see table). (It is advisable to use the same batch of TAE in the tank as was used in the gel)
11. Mix 5 volumes of PCR product with 1 volume of 6x gel loading buffer
12. Load samples into the wells in the gel
13. Load 1kb ladder into at least one well in each row
14. Run the gel at the appropriate voltage for about 30 minutes (see table)
15. Photograph the gel under ultraviolet light


MINI MIDI
1xTAE in gel 25ml 100ml
Agarose 0.2g 0.8g
Ethidium
Bromide 1ml 4ml
1xTAE
in tank 250ml 700ml
Voltage 75V 150V
1xTAE

1. Add 1 volume of 50xTAE to 49 volumes of water
2. Store at room temperature

50xTAE

1. To a 1 litre container add 242g of Tris Base
2. Add 57.1ml glacial acetic acid
3. Add 100ml O.5M EDTA pH8.0
4. Make up to 1 litre with water
5. Store at room temperature

6x Gel Loading Buffer

1. To 7 volumes of water add 3 volumes of glycerol
2. Add 0.25% bromophenol blue (runs at about the same rate as linear double stranded DNA of 300bp)
3. Add 0.25% xylene cyanol FF (runs at about the same rate as linear double stranded DNA of 4kb)
4. Store at +4C
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  #2  
Old 01-04-2007, 03:51 PM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Thanks for the detail protocal.
I have a question; in 25ml agarose gel, you add EtBr 1ml, what is the concentration of EtBr in 1 ml?
Thanks.
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Old 01-05-2007, 02:39 PM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Hi,

we use 1 microliter to 50ml agarose (in 1x TAE) from 25mg/ml stock.

Cheers
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Old 03-10-2007, 03:21 AM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

whitesnow,

for standard electrophoresis runs, we use EtBr stock of 10ug/ul
for 100 ml agarose gel, we use 5ul of EtBr stock in order to get a final concentration of 0.5ug/ul of EtBr.
I hope it is now clear to you

cheers!!!

rakhi
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Old 05-23-2008, 04:42 PM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

About that Ethiudium...
I always use 2ul of 10ug/ul ethidium bromide stock per 100ml gel.
This way, the background is always darker, which increases my signal-to-noise ratio... Recommended!

Last edited by admin; 02-15-2011 at 11:04 AM.
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  #6  
Old 02-15-2011, 04:55 AM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Thanks for that . I wondered how I could do it and the protocol is just what I needed
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  #7  
Old 06-08-2011, 05:19 AM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Quote:
Originally Posted by Whitesnow View Post
Thanks for the detail protocal.
I have a question; in 25ml agarose gel, you add EtBr 1ml, what is the concentration of EtBr in 1 ml?
Thanks.
You can buy it in 10mg/ml concentration but this is too much.
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  #8  
Old 06-20-2011, 10:50 PM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Thanks for your replies rhabdo,
I've been busy these couples of week, so I didn't response to your answers. Yes, I am looking at the soil microbiota as a whole rather than isolating specific bacterial species. In my DGGE profile the intensities of each band is about the same at each lane
(seen at the same row), that's what make my head aches. I was hoping to see a different profile lane to lane, but it seems they were all look the same! But from one band to another in the same lane/column have different intensities. I used 338F-GC and 518R. Any ideas how can I avoid two bacterial species from stopping at the same point in my DGGE gel beside using different primers?
Thanks for your kind answer...
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Old 06-20-2011, 10:53 PM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

Hi, sorry to bother you all, but maybe you can lend me a hand for my problem

I am looking at the soil microbiota as a whole rather than isolating specific bacterial species. In my DGGE profile the intensities of each band is about the same at each lane
(seen at the same row), that's what make my head aches. I was hoping to see a different profile lane to lane, but it seems they were all look the same! But from one band to another in the same lane/column have different intensities. I used 338F-GC and 518R. Any ideas how can I avoid two bacterial species from stopping at the same point in my DGGE gel beside using different primers?
Thanks for your kind answer...
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  #10  
Old 12-10-2011, 05:23 AM
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Default Re: Agarose gel electrophoresis of PCR products - Method and Protocol

dear sir thank u very much but i have to see the tables

mahmoud manama
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