Aug 24, 2022
Protocol for DNA Extraction from Tepary Bean
- 1Boyce Thompson Institute
Protocol Citation: Aparna Srinivasan, Magdalena M Julkowska 2022. Protocol for DNA Extraction from Tepary Bean. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpb78plzp/v1
Manuscript citation:
Semagn K. Leaf tissue sampling and DNA extraction protocols. Methods Mol Biol. 2014;1115:53-67. doi: 10.1007/978-1-62703-767-9_3. PMID: 24415469.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2022
Last Modified: August 24, 2022
Protocol Integer ID: 68013
Keywords: DNA Extraction, CTAB, Tepary bean
Abstract
Protocol to extract high quality DNA from Tepary Bean leaves using modified CTAB method.
Materials
Buffer and Chemicals Required
1. CTAB Buffer pH.8
- CTAB- 20 g/L
- Tris- 12.11 g/L
- EDTA- 7.44 g/L
- NaCl - 81.89 g/L
2. RNAse A (10mg/ml)- Dissolve in TNE buffer
- Tris-cl - 10mM
- Nacl - 15 mM
- EDTA - 1 mM
- Reconstitution as per Sigma Aldrich Roche Document (Product code: 10109142001)
RNase A can be dissolved at a concentration of 1 to 10 mg/ml in 10 mM Tris-HCl, pH 7.5, 15 mM NaCl, heated to
100 °C for 15 minutes to inactivate contaminating DNases and cooled slowly to room temperature and dispend
into aliquots. Roche recommends subsequent storage at -15 to -25 °C.
3. Proteinase K (20mg/ml) : Dissolve in TE buffer
4. Phenol:Chloroform kit - Stored at 4 °C
5. Isopropanol
6. 3M Sodium Acetate
7. 70 % Ethanol (Ice-cold)
8. TE Buffer pH.8
- Tris- 10mM
- EDTA- 1mM
9. Agarose
10. Ethidium Bromide
11.Size Marker: 1 kb DNA ladder
Instrument
1. Centrifuge
2. Hot Water Bath
3. Spectrophotometer- Nanodrop
4. Gel Documentation system
Collection of leaf sample
Collection of leaf sample
Collect 100 mg of young leaf from Tepary Bean plants in a 2ml eppendorf tubes containing two sterile glass beads and freeze immediately in liquid nitrogen.
DNA Extraction
DNA Extraction
Add 600 μl of pre-warmed CTAB buffer and proteinase K (20 mg/ml), invert the tubes gently to mix together and incubate the tubes in water bath at 65 °C for 30 min.
Invert the tubes once in every 10 min to homogenize the tissue and buffer.
CTAB buffer added to sample- clear green solution
After 30 min, remove the tubes from water bath and centrifuge at 14000 rpm for 15 min.
Transfer cleared lysate of 500 μl to a new 1.5 ml tube. Add RNase (10 mg/ml) and keep at 37 °C for 30 min.
Add equal volume of Phenol: Chloroform (phenol:chloroform kit), and vortex well.
Phenol:Chloroform mixed with sample
Centrifuge at 14000 rpm for 15 min. Transfer the upper aqueous layer to a new 1.5 ml Ep tube.
Transfer upper aqueous layer to a new tube
Do not touch below layers while transferring the aqueous phase.
Repeat the Phenol: Chloroform alcohol step if the aqueous phase is not clear.
Add equal volume of isopropanol, and mix very gently, keep for few minutes to precipitate nucleic acid.
White strands noticed after adding isopropanol
Centrifuge at 14000 rpm for 10 min.
Pellet can be seen at the bottom.
Discard the supernatant.
Add 300 μl of 3M Sodium Acetate and 500 μl 70 % ethanol and centrifuge at 14000 rpm for 5 min.
Discard the supernatant.
Add 200 μl 70 % Ethanol and centrifuge at 14000 rpm for 5 min.
Discard the supernatant.
Air-dry the pellet, dissolve the pellet in 50 μl TE Buffer.
DNA is run on a 0.8% Agarose gel to check whether it is degraded or having RNA contamination.
If RNA is present, treat the samples with RNAse A and precipitate again from phenol:chloroform step 4.
Nucleic acid concentration is measured in Nanodrop, and Ratio of A260/A230 with 1.8-2.0 indicates purity of DNA.
In case the values are <1.8, it indicates contamination such as carbohydrate or phenol.
0.8 % Agarose Gel picture
Store DNA sample at -80 °C until use.