Rahul Arvind Jamdade Sir is there any other method using Tris CL and KCl as lysis buffer and refrigerating in LN2?
We use a salting-out isolation. The first step is to get a good tissue homogenized using a Potter-Elvehjem, for which it is very useful in a cell lysis with a high concentration of proteinase K. Next overnight in cell lysis at 56 ° C. You can then continue the process of salting-out.
Butterflies legs are good for sampling because of as a conservation biologist we could not take a part of the animals insured of that even a single legs are not caused whole animals thats why we make it sampling only for legs
You can use this method..................
Simple method for the isolation of DNA (up to 25 µg) from fresh, frozen or stored insect and
arthropod specimens in as little as 15 minutes.
The organic denaturant/proteinase K-free procedure is ideal for processing small organisms
including mosquitoes, bees, lice, ticks, and D. melanogaster.
State-of-the-art, ultra-high density BashingBeads™ are fracture resistant, chemically inert and can
be used with any bead mill, disrupter, or vortex that can accommodate standard 2 ml tubes.
Also, compatible with mammalian tissues, cultured cells, and whole blood.
from Zymo RESEARCH Carporation..
Hi! In our lab we use the method of TNES, described in
MARTINS, J.; SOLOMON, S. E.; MIKHEYEV, A. S.; MUELLER, U. G.; ORTIZ, A.; BACCI, M. Nuclear mitochondrial-like sequences in ants: evidence from Atta cephalotes (Formicidae: Attini). Insect Mol. Biol., v. 16, n. 6, p. 777-784, dez. 2007.
We work with ants, but I've used this method in Molecular Biology classes and it is very good for a lot of other organisms including many arthopods, fungus, vertebrate tissues, bacteria...
We also use the kit Invisorb Spin Tissue Mini Kit (Invitek).
It is important to know what you want the DNA for. For sequencing long segments of DNA, Chelex is not recommended because it produces more DNA fragments. Chelex is more suitable for microsatellite typing. While searching an alternative I found the HOTShot technique. I actually switched from Chelex to the HOTShot method for 2 reasons: 1) HOTShot is far cheaper and easier to do than Chelex and 2) HOTShot is suitable for thae amplification of longer sequences. We have successfully extracted DNA from large (like bees), medium sized (like flies) and very small (trips) insects with the hotshot method. Just google "HotShot DNA" and you will get it.
It is better to use the parts of the insect which have more muscle, like legs and thorax. The amount of tissue depends on your particular requirements and availability of specimens (and their size). We use entire individuals when extracting DNA from trips, but only the tip of a leg when analyzing bees.
Hello,
This protocol is for isolating dna from insect tissue preferably legs, head and wings. We are using this protocol with sucess rate of 80%. Kindly cite at (dx.doi.org/10.17504/protocols.io.bfajjicn)
* Successful and inexpensive DNA extraction protocol from insects with low amount of tissue*
Considering that the insect to be used for DNA extraction was preserved/collected in Absolute Alcohol (Molecular grade). Dry the sample and transfer into the new microcentrifuge tube.
1. Add pre-warmed CTAB (600ul) (65°C) +3ul of Beta-Mercaptoethanol+ 10ul of 20% SDS
2. Crush the sample and then add 3ul of Proteinase-K.
3. Vortex each tube for 3-5 minutes vigorously.
4. Incubate at 65 °C for 4-5 hours (prefer shaking heating block), or vortex it at the interval of 30 minutes if possible.
5. After incubation, cool the samples to room temperature, and centrifuge at 14,000 rpm for 10 min
6. Add equal volume of Phenol: chloroform: Isoamyl alcohol (25:24:1).
7. Vortex for 3-5 minutes and then centrifuged at 14,000 rpm for 10 minutes.
8. Take supernatant in fresh microfuge tube and discard the pellet.
9. Add Chloroform: Isoamyl alcohol (24:1), 600ul in each tube and mix it (vortex).
10. Centrifuge at 12,000 rpm for 10 minutes.
11. Take the supernatant in fresh microcentrifuge tube.
12. Add chilled isopropanol (400 ul) and mixed slowly until white flakes appear.
13. Then keep it at -20°C or -50°C in deep freeze for 1hour.
14. Bring the sample at room temperature and then centrifuge at 10,000 rpm for 10 minutes.
15. Decant the supernatant and add 70% chilled ethanol (400ul) + ammonium acetate (100 ul) to the pellet for washing.
16. Centrifuge at 10,000 rpm for 10 minutes.
17. Decant the supernatant carefully; add 400ul of absolute alcohol and Centrifuge at 10,000 rpm for 10 minutes.
18. Decant the supernatant and dry the pellet at room temperature or in speed vac.
Speed vac program: Program name ‘-OH’, for 15 minutes at 30°C.
19. Add nuclease free water (10-30ul) and incubate samples in a dry bath at 55 °C for 30 minutes.
20. Keep/store at -20°C or -80°C (long term storage)
CTAB EXTRACTION BUFFER – (For 50ml)
1M Tris-HCl 5ml, 0.5 M EDTA 2ml, 5M NaCl 17.5ml, 10% CTAB 10ml (5 gm in 50 ml), Distilled Water 15.5 ml to make up the volume. Set the pH to 7.5-8.0. (Autoclave the whole content before use.)
@rahul, sir kindly if you could tell the exact amount, that how much quantity of ammonuim acetate was used in step 13? and the amount of chemicals used in step 6 and 12?
thanking you in anticipation..
@Rahul Jamdade
Do you have a reference for that protocol I could get?
Hello! Do you already have a preferred protocol ? I am working with Agaonids, fig wasps, they are very tiny!
@Rahul Jamdade, I will try your protocol, but at what temperature do you incubate overnight ? Thank you very much !
How can i isolation of DNA of corn aphid, Rhopalosiphum maidis? Please give me protocol about this insect.
Hola.
CUanto volumen se agrega de fenol: cloroformo: isoamlico en el paso 6?
Hi Sir,
Could you please tell the steps involved in drying the mite samples dipped in ethanol?
Rahul Arvind Jamdade
Hello,
Commercial kits give good results and are safe and easy to handle. I used Qiagen Blood and Tissue kit to extract DNA from dipteran samples stored in 70% ethanol for years which gave me satisfactory results.
Paylaşım için öncelikle teşekürler efendim. Bu protokol için alabileceğim bir referans var mı?
DNA isolation from Pink Bollworm
Crush insect in 200ul of ice-cold homogenization buffer. Add 20ul lysis buffer keep at room temperature and incubate for 20min. After place tubes at 65oc for about 45 mins. Add 5ul of RNase to each tube incubate for 30min. Add 50ul of potassium acetate incubate at 4oc for 30min. Add equal volume of Chloroform:Phenol mix by inverting. Centrifuge at 12000rpm for 10 min. Take aqueous layer and add equal volume of Isopropanol and incubate at -20oc for overnight. Centrifuge at 12000rpm for 10min discard supernatant and wash pallet twice with 70% alcohol. Air dry pallet and dissolve in 50ul of TE buffer.
· Homogenisation buffer pH 8
0.1M NaCl
0.2M Sucrose
0.01M EDTA
0.03M Tris HCL
· Lysis buffer pH 9.2
0.025M EDTA
2.5% SDS
0.5M Tris
8M Potassium acetate
· RNase 1mg/ml
The phenol-chloroform isoamyl DNA extraction method can be more resilient towards the removal of pigments and contaminants.
There are kits available designed for certain insect types. However, there may still be optimisation involved on either side.
I suggest that you have look at using liquid nitrogen -pestle and mortar grinding of your insect sample to expose the maximum amount of tissue you can. Be gentle, this can damage DNA if done excessively.
Also, done heat SDS, it is not thermostable and degrades quickly. check MSDS
Rahul Arvind Jamdade Sir CTAB method can be used to isolate DNA from ixodid ticks and mosquitoes.
Rahul Arvind Jamdade Sir is there any other method using Tris CL and KCl as lysis buffer and refrigerating in LN2?
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