DNA isolation protocol from Bacteria

 Short note on DNA isolation protocol from Bacteria concept by the biotechnify 

Abstract

This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.

What is genomic DNA in bacteria?

Most bacteria have a genome that consists of a single DNA molecule (i.e., one chromosome) that is several million base pairs in size and is "circular" (doesn't have ends like chromosomes of eukaryotic organisms).

What is the principle of DNA extraction?

The DNA extraction process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure DNA. The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.

Materials and Reagents

1. Tris base (Calbiochem-Behring)
   
2. Proteinase K (Sigma-Aldrich)
   
3. Phenol\chloroform (1: 1) (EM Science)
   
4. 200 proof ethanol (Pharmco-AAPER)
   
5. RNAase (Life Technologies, Invitrogen™)
   
6. Ethanol
   
7. SDS
   
8. EDTA
   
9. Tryptone
10. Yeast extract
11. NaCl
    
12. LB medium 
    
13. TE buffer 

Equipment

1. Tabletop centrifuge (Eppendorf)
   
2. 1.5 ml Eppendorf tube
   
3. Incubator
   
4. Gloves

ADDITIONAL Equipment :

• Waterbath or beaker and thermometer

• Mini Centrifuge

• Agarose Gel Equipment (optional)

• DNA Loading Buffer (optional)

• 70% Ethanol (Optional)

Procedure

1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
   
2. Discard the supernatant.
   Note: Remove as much of the supernatant as you can without disturbing the cell pellet.
   
3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.
   
4. Incubate 1 h at 37 °C.
   
5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
   Note: Do not vertex the tube—it can shear the DNA.
   
6. CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.
   
7. Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
   
8. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
   
9. Steps 4-6 can be repeated until the white protein layer disappears.
   
10. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
    
11. Spin at max speed for 5 min.
    
12. Remove aqueous layer to new tube.
    
13. To precipitate the DNA, add 2.5 or 3 volume of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
    Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see DNA pellet. It looks transparency when it is wet and turns to white when it becomes dry.
    
14. Incubate the tube at -20 °C for 30 min or more.
    
15. Spin at max speed for 15 min at 4 °C.
    
16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
    
17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
    
18. Resuspend DNA in TE buffer.
    Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.
19. Check isolated Gemonic DNA on an agarose gel.
    Note: we expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded.
    

Precautions :

 Material finely ground in liquid nitrogen should be immediately transferred into

the extraction buffer and must not be allowed to ‘sweat’..

 In chloroform : isoamyl alcohol extraction, the aqueous phase should be

carefully removed and organic phase re-extracted to ensure full recovery of

DNA. If no separation is observed between the two phases, may be due to high

concentration of DNA and /or cell debris in aqueous phase, dilution with more

digestion buffer and re-extraction is the solution.

 Care should be taken to do the operations as gently as possible. Vortexing,

pipetting using fine tips etc. should be avoided to prevent the shearing of DNA.

 DNA should not be over dried as resuspesion in TE become difficult.

 All the glassware, plastic ware, pestles and mortars etc. should be

decontaminated properly. Care should be taken to prevent cross-contamination.

 Blank extraction controls are carried out along with normal extractions to check

for any contamination.

TIME Required :

• Day 1: 2 hours

📲End. DNA isolation protocol from Bacteria