Conformation Sensitive Gel Electrophoresis CSGE protocol

i am using the same protocol my product size is 350 bp,running voltage is 350 volts for 16 hrs i am not getting good results bands are not very clear
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how can we use csge for hemophilia dna analysis
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some of the are confusing...

what is "BAP 40% 15% 60ml"
stock final volume?
99:1Acrylamide: BAP 40% 15% 60ml
20X TTE (Glycerol Tolerant) 20X 0.5X 4ml
Ultra Pure Formamide 15% 24ml
Ethylene Glycol 10% 16ml
Sterile Water 54.4ml
10%AmmoniumPersulfateSolution 0.1% 1.6ml?

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hello there . So far so good but i was wondering if you can help me finding the mechanism of BAP in enhancing the polymerization of the gel.
thanks again
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Conformation Sensitive Gel Electrophoresis CSGE gel Protocols:

Stock Final Volume
99:1Acrylamide: BAP 40% 15% 60ml
20X TTE (Glycerol Tolerant) 20X 0.5X 4ml
Ultra Pure Formamide 15% 24ml
Ethylene Glycol 10% 16ml
Sterile Water 54.4ml
10%AmmoniumPersulfateSolution 0.1% 1.6ml
(Fresh prepared APS)

TEMED 0.07% 110ul


Procedure:
1.Mix every reagent except 10% APS and TEMED, and leave mix at room temperature .
Add the last two reagents immediately before pouring the gel. pour gel and insert comb.
Allow mix to polymerize for at least 1 hour at room temperature.

Sample Preparation:

1.Perform PCR under normal optimized conditions.

2.After amplification, mix equal amounts of wild type product and each sample product, then denature at 95°C for 5 min and anneal at 68°C for 30-60 min. This step encourages heteroduplex formation. From 2-15m(50-100ng) is used for analysis by CSGE.

3.For each 10ml mixed product, add 2ml 10X loading dye (10X BlueJuice Gel Loading Buffer Invitrogen Cat. No. 10816-015) including 40% sucrose, 0.25% xylene cyanol, 0.25% bromophenol blue in dH2O)




Running Conditions:

1.For 1L buffer: 25ml 20 X TTE + 975ml dH2O. Use 0.5X TTE as a running buffer (USB, cat. no.US75827)

2.Carefully remove the comb and bulldog clips. Immediately rinse wells with syringe before loading.

3.Load~6ml of sample and dye to each well.

4.Run 6-8 hours at 40Watts. Or overnight at 400 Volts.

Gel Staining:

1.Make ethidium bromide solution: 20ml of EtBr (10 mg/ml) in 100ml of 0.5 X TTE. Pour solution over gel only; Let stain for 6 min.

2.De-stain for 10 min. Rinse with 0.5X TTE.

3.The xylene cyanol dye in the loading buffer runs about the same as ~200 bp DNA strand.

4.Use UV light to see the bands, blot the gel with Whatman paper, cut off the extra gel, and take to imaging system. To release the gel from Whatman paper, pour water on the back of the paper and lift the Whatman paper off.






Notes:

1.During storage, acrylamide and bisacrylamide are slowly converted to acrylic acid and bisacrylic acid. This deamination reaction is catalyzed by light and alkali. Store the solution in dark bottles at 4°C. Fresh solutions should be prepared every few months.

2.Gels may be stored for 1 day before they are used. After polymerization is complete, surround the comb and top of the gel with wet tissue and store at 4°C.

3.Wash out the wells thoroughly comb is removed before loading samples. small amounts of acrylamide solution trapped by the comb will polymerize in the wells, avoid producing irregularly bands of DNA.
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hOW TO USE THE FORMAMID AS A LODING BUFFER
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how to use the formamide as mild denaturing function inside the CSGE method,
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