TITLE: Protocol for DNA Extraction from Cowpea
AUTHORS: Aparna Srinivasan, Magdalena M Julkowska

[DESCRIPTION]
Protocol to extract high quality DNA from Cowpea leaves using modified CTAB method.

[STEPS]
SECTION: Collection of leaf sample
1. 
Collect 1 fully expanded leaf from Cowpea plants in a 50ml centrifuge tube containing five sterile glass beads (5mm diameter) and freeze immediately in liquid nitrogen.
SECTION: DNA Extraction
7. 
Add equal volume of Chloroform, and vortex well.
 
 

Centrifuge at 14000 rpm for 15 min. Transfer the upper aqueous layer to a new 1.5 ml Ep tube (~350-400 uL)
 

Do not touch below layers while transferring the aqueous phase. 

Repeat the Phenol: Chloroform alcohol step if the aqueous phase is not clear.

SECTION: DNA Extraction
8. 
Add equal volume of isopropanol, and mix very gently, keep for few minutes to precipitate nucleic acid.


Note: It may take up to 30 minutes of incubation at room temperature to observe precipitate nucleic acid
 
 
Centrifuge at 14000 rpm for 10 min. 

Pellet can be seen at the bottom. 

Discard the supernatant.
SECTION: DNA Extraction
6. 
After 30 min, remove the tubes from water bath and centrifuge at 14000 rpm for 15 min to ensure lysate is cleared.

Transfer cleared lysate of 500 μl to a new 1.5 ml tube. Add 10 ul of RNase (10 mg/ml) and keep at 37 °C for 30 min.
SECTION: DNA Extraction
9. 
Add 300 μl of 3M Sodium Acetate and 500 μl 70 % ethanol and centrifuge at 14000 rpm for 5 min.

Discard the supernatant.
SECTION: DNA Extraction
10. 
Add 200 μl 70 % Ethanol and centrifuge at 14000 rpm for 5 min.

Discard the supernatant.
SECTION: DNA Extraction
11. 
Air-dry the pellet, dissolve the pellet in 50 μl TE Buffer.

SECTION: DNA Extraction
5. 
Add 600 μl of pre-warmed CTAB buffer and 12 ul of 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.

 
13. 
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.

 
14. 
Store DNA sample at -80 °C until use.
SECTION: Collection of leaf sample
2. Grind tissue using vortex, ensuring tissue remains frozen. Transfer ~.5 mL of ground tissue to a new 2 mL eppendorf tube containing two 2mm glass beads.
SECTION: DNA Extraction
4. Before beginning: 
Heat water bath to 65 °C and place CTAB buffer in 65 °C water bath for 20 minutes
Heat incubator to 37 °C
Place 70% ethanol in 4 °C 
Make sure RNAse (10mg/mL) is already made and treated
Make sure proteinase K (20mg/mL) is made

*Note on CTAB buffer: Because of high phenol content in the leaf tissue, it may be helpful to add 1% PVP to the CTAB buffer  
SECTION: Collection of leaf sample
3. Further beat the tissue by placing in the bead beater for 1 minute in one direction, then 1 minute in the opposite direction. 
SECTION: DNA Extraction
12. Place at 4°C overnight, then measure concentration and purity on a Nanodrop