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Page | 57 Extraction and isolation of DNA
Pre DNA extraction
Takeout stored moth legs in a cavity block and remove the scale by rubbing gently with very fine Camlin brush (000’). Transfer the legs to another cavity block containing distilled water and wash. Washed legs are placed on tissue paper for drying.
DNA Extraction:
There are commercially available kits for extraction of insect genomic DNA. All the kits come with extraction protocols. However, traditional method of DNA extraction is useful for the beginners. The Cetyl trimethylammonium bromide (CTAB) procedure for isolating DNA was simplified from one proposed by Doyle and Doyle (1990). The steps are as follows:
i. All six legs of adult moths were taken in a 1.5 ml eppendorf tube and powdered well using liquid nitrogen.
ii. The ground tissue was transferred to a 50 ml sterile centrifuge tube by adding 15ml of CTAB buffer (Extraction buffer- 2%C-TAB, 1.4 M NaCl, 20 mM EDTA (Disodium) and 10mM Trisbase (pH 8)). Then 50 L of - mercaptoethanol was added to each tube and the extract was mixed well by inverting for two min. The tubes were incubated in a water bath maintained at 650C for an hour with constant stirring at an interval of 15 min.
iii. After an hour, 15 ml of chloroform isoamyl alcohol (24:1) was added to the incubated sample and mixed well by inverting. The tubes were then centrifuged at 10,000 rpm for 20 min.
iv. The aqueous upper phase was carefully transferred using one ml tips into fresh sterile centrifuge tubes. To this supernatant, 0.7 volumes (10.5 ml) of cold isopropanol were added.
v. The tubes were carefully inverted and kept for 5 min on ice. The precipitated DNA was then centrifuged at 6000 rpm for 20 min and sedimentation of DNA as a pellet was seen.
vi. Further the supernatant was decanted gently and the tubes were inverted on a clean filter paper.
vii. The pellet was washed twice by suspending in one ml of 70% ethanol for 5 to 10 min and centrifuged at 6000 rpm for two min.
viii. Ethanol was drained off slowly and the pellet was vacuum dried in a dessicator for 5 to 10 min. The pellet was then dissolved in 500 L of TE buffer by flicking the tubes. TE buffer = 0.1mM Tris + 0.05mM EDTA
Page | 58 ix. To remove the RNA, 5L of RNase (10mg/ml) was added into the DNA solution and
incubated at 37oC in a water bath for one hour.
x. Again the DNA solution was cleaned by washing with equal volume (500 l) of phenol:
choloroform: Isoamyl alcohol (25:24:1) by invert mixing several times and centrifuging at 6000 rpm for 15 min to separate the two phases.
xi. The aqueous upper phase was transferred into a clean 1.5 ml eppendorf tube and two volumes of absolute ethanol were added to precipitate the DNA.
xii. The pellet was washed with 70% ethanol twice and dissolved in 500 l TE. The extracted DNA was quantified using both Nanodrop DNA quantifier and also by electrophoresis on 0.8% agarose gel.
DNA purity assessment
Two μl of isolated DNA were diluted to one ml with TE buffer and the absorbance at 260 and 280 nm were recorded against a buffer blank. A 260/280 nm ratio for all the samples was calculated to check the purity. DNA was quantified using the formula:
μg ds DNA/μl = (A260 * 40)/2
Further all the samples were diluted to a final concentration of 10 ng/μl.
DNA quantification on agarose gel
Agarose gel submersible electrophoresis is used to separate the DNA based on size (0.1 to 2.5 kb), in a submerged horizontal tray. The tray was chosen in such a way that its length allowed the required band resolution. Genomic DNA electrophoresed at 1% agarose concentration.
Preparation of agarose gel
i. One g of agarose was weighed and taken in a clean 250 ml conical flask. To this 100 ml of 1x TBE buffer was added. [TBE buffer (0.89M Tris base, 0.02 MEDTA, 0.89M Boric acid) pH = 8].
ii. Agarose was dissolved by heating at 100ºC for 15 min. After agarose completely melts, it was cooled to 60°C and 3.5 μl of ethidium bromide was added (10 mg/ml) and mixed.
iii. The ends of gel casting trays were sealed with cellophane tapes. Agarose was poured;
comb was inserted and allowed to solidify. After solidification the tape on either side was removed and the gel was immersed in electrophoresis tank containing 1x TBE buffer. Then the comb was removed.
iv. To 5 μl of DNA samples 3μl of 6x loading buffer was added, mixed well and loaded into the well. 3μl of standard uncut Lambda DNA was used as marker. (Loading buffer or tracking dye 6x – 40% sucrose, 0.025% bromophenol blue, 0.25% xylene cyanol).
Page | 59 v. Electrophoresis was carried out at 50V for 2 to 3 hours until the bromophenol blue dye migrated two-thirds of the gel. The gel tray was removed and the gel was observed under UV transilluminator and documented using Gel Doc system. The quantity of the DNA was determined based on the intensity of the band relative to lambda uncut band.
Primers used
Mitochondrial cytochrome oxidase sununit I gene (mtCOI gene) was amplified using the forward primer LCO1490 and reverse primer HCO2198, developed by Folmer et al. (1994).
Name of the primer Sequence (5’ to 3’)
LCO1490 5' GGT CAA CAA ATC ATA AAG ATA TTG G 3'
HCO2198 5' TAA ACT TCA GGG TGA CCA AAA AAT CA 3'
PCR amplification
The reaction mixture was set up in sterile 0.2 ml microfuge tubes. The reaction mixture volume per reaction was as follows:
Reaction Mixture Quantity (μl)
10x Taq Polymerase buffer 5
2 mM dNTPs 5
MgCl2 (2mM) 1
Forward primer (5pMole/μl) 2
Reverse primer (5pMole/μl) 2
Taq Polymerase (1U) 0.3
Template DNA (20-25ng) 4
Sterile water 30.7
Total volume 50.0
Reaction mixture was vortexed and centrifuged. Amplifications were performed using master cycler with following temperature transitions:
Steps Temperature (ºC) Time
Initial denaturation 94 5 min
Denaturation 94 1 min
Annealing 52 30 sec
Elongation 72 1 min
Extension 72 5 min
Page | 60 Thermal cycle was programmed for 35 cycles with one cycle of initial denaturation and steps 2-4 were repeated 35 times.
Agarose gel electrophoresis of PCR product
The PCR products were resolved by electrophoresis using 3 percent agarose gel in1X Tris borate EDTA buffer for about 2 hours at 110V along with 100 bp ladder. The gel was stained with ethidium bromide (0.5 μg/ml), viewed under UV Tran-illuminator and photographed immediately for further interpretation using Gel-Doc system.
Sequencing
PCR products are purified and dissolved in 0.1’ TE by gel extraction/PCR cleanup methods.
Purified samples are sequenced at the specific commercial facilities.
Sequence analysis
Obtained DNA sequences were proofread by eye and aligned using MEGA version 4.1 (Tamura et al., 2007).
Sequences were then checked for plausibility using BLAST with the blastn algorithm (Altschul et al. 1990; URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi) as well as the BOLD Identification System (IDS, URL: http://www.boldsystems.org/index.php/IDS_OpenIdEngine)
The sequence details are analyzed carefully, submitted to NCBI for GenBank Accessions and subsequently uploaded to BOLD. All generated sequences, together with photographs and collection details, have been deposited at the Barcoding of Life Database (BOLD;
www.boldsystems.org) under the specific project code. DNA barcoding analyses will be done through the online interface of the BoLD website. The taxon identification tree was based on the Kimura 2-parameter distance model (Kimura, 1980), with the filter set to sequences with length >100 basepairs, and all codon positions included.
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