Hello... i extract gDNA from mice (and chickens) and you could use a regular lysis buffer, treat with RNase A and Proteinase K and phenol:chloroform extraction. We use this for some protocols

Chelex-100 also works very well, but you have to crash them before with a homogenizer or with a pipette tip and allow an overnight step in a lysis buffer, then proceed with the gDNA extraction with chelex.

Salut Mamadou

j espre ca t aidera

 si tu as autre difficultes laisse moi savoir

Protocol

1. Preparatory Dissection of Mosquito Specimens

Place mosquito on a small cutting of parafilm and mount on dissecting microscope.

Cover in drop of deionized water to soften tissue.

Incise the mosquito carcass precisely at the joint between the thorax and abdomen, thus nicking off the head and thorax from the abdominal section.

Transfer each dissected mosquito section into a clean autoclaved 1.5 ml microcentrifuge tube prelabeled with mosquito ID number details and appropriately marked to denote body section (e.g."m" for mid-gut section; "ht" for head and thorax section).

Discard parafilm and carefully wipe microscope stage with 70% alcohol-moistened Kimwipe before processing the next mosquito.

2. DNA Extraction from Mosquito Specimens

Pipette 20 μl of deionized water into sample tube and use pipette tip to grind the submerged mosquito section into a uniform suspension.

Add 100 μl of autoclaved 1X PBS/1% saponin solution to sample homogenate and mix by gentle momentary vortexing.

Incubate at room temperature for 20 min.

Centrifuge at 20,000 x g for 2 min and discard supernatant.

Resuspend pellet in 100 μl 1X PBS.

Centrifuge again at 20,000 x g for 2 min and discard supernatant.

By gentle vortexing (5 sec), resuspend pellet in 75 μl sterile deionized water and 25 μl of 20% w/v Chelex-100 resin suspension in deionized water.

Pierce hole in lid of sample tube using sterile 23G hypodermic needle flamed in Bunsen burner.

Boil sample suspension in water bath on floating rack (or in heating block) for 10 min.

Centrifuge at 20,000 x g, for 1 min and transfer ensuing DNA solution into prelabeled storage vial for use as template in PCR applications.

Hello Monsieur Tambo,

Thanks a lot for your help,

I saw that your method is the most academic. But was it effective? Have you ever tested it? How was your DNA purity? Can I work directly with mosquito leg or it's better to prepare the sample by the method?

About the w/v Chelex-100, please can you redirect me to the manufacturer you used to purchase the product.

 Miss Ejimedo Madogwe, I extract the DNA from mosquito sample, do you think that your method is accurate for my sample?

Dear Giacomo Maria Paganotti, Do you have any details about your protocol step please? Its look like that your's is near from one proposed here and it seems to be really interesting. Can you share the protocol please?

Mamadou make a test run on DNA extraction ad test the DNA quality and purity and adapt to your context. The protocol is working very well but the final genomic DNA depends on the below factors.

 NOTE: I can't take a new protocol or SOP and implement without prior VALIDATION. This is a basic requirement in Scientific research method( experimentation and validation/standardization of protocol).

The quality and purity of the obtained genomic DNA depends on many factors , including the source of your sample, the duration of the field, the storage conditions, reagents and read on previous literature for basics on PCR or genotyping.

Best wishes

Dr Tambo

Hello Mamadou, yes i believe my method will work with your experiment (it should work with any tissue sample really)... However, i Saw Dr Tambo's response. i also use chelex to extract genomic DNA for genotyping experiments. it works really well. And i believe it is a much cheaper alternative.it is also your best bet since he has apparently used this protocol on mosquitoes.

Nevertheless, if you want details of my protocol, i will be happy to share.

Thank you Dr Tambo, I Will implement my method based on your suggestion. Indeed, validation before using a routine application is unavoidable. 

Ejimedo, I would love to get your protocol, it doesn't cost nothing to read it. You can use my mail or share it here ([email protected]). Thx again

If your lab can go for QIAamp DNA extraction kit ( QIAGEN or INVITROGEN) that is the best.

Yes I know and you're right, but my wallet is too short! To much sample to exploit

This is the protocol for genomic DNA extraction from mice tissue samples for qPCR.

Lysis

1.5ml

75ul of 20% SDS

30ul of 0.5M EDTA

75ul of 1M Tris pH 8.1

1290ul of milliQ

Add PIC and PMSF to Lysis buffer

-Add 300ul of Lysis buffer per sample

-Incubate on ice for 30mins

-Treat with RNase A (20ug) for 30mins at 37˚C (2ul of stock 10mg/ml)

-Treat with 100ug of Proteinase K for 1-2hours at 37˚C (5ul of 20mg/ml stock)

For this protocol, I usually use the Qiagen purification kit as I use these samples for quantitative PCR.

This is the genotyping protocol we use on mice:

DNA Extraction

Make 10% Chelex extraction buffer

To make 50ml

5g chelex

500ul of 20% SDS

500ul of  1M Tris pH 8.0

50ul of 0.5M EDTA pH 8.0

47.5ml of milliQ water

§  For each sample, you will need 250ul of 10% chelex extraction solution and 2ul of Proteinase K (20mg/ml).

§  Chelex extraction solution may be stored at room temperature but proteinase K MUST be added fresh each time. Therefore, only add it to the quantity you need.

For example, if you have 5 mice, you need

5 X 250ul of 10% chelex solution=1250ul

+

 5 x 2ul of PK=10ul

so make 1250ul and add 250ul  to each sample.

§  Incubate overnight at 55˚C (at 15000rpm).

Day 2

o   Boil the samples for 5minutes at 95˚C t inactivate Proteinase K

o   Centrifuge at 13000rpm for 10minutes

o   The DNA is in the supernatant.

Use immediately or store at 4˚ C.