I am trying to extract DNA from a Siphoviridae phage.... according to many times phage DNA extraction and check on electriophoresis gel the geneome size should be 20 kb.
protocol I had followed:
1. liquid culture of bacteriophage up to 10^8
2. 10% PEG treatment for over night stirring at 4C.
3. Centriufigation for 1 hour at 10000rpm
4. pellets were resuspended in PBS buffer.
5. cscl gradient for 2 hours at 37000 rpm.
6. band pick up and band were cell downs for 3 hours in PBS buffer at 37000 rpm and pellet were resuspended in 500 ul of pbs buffer.
7 dnase and rnase treatment to remove any host genome
8.proteinase k treatment for 1 hour at 60C
9. phenol choloroform extraction of bacteriophages for three times
10.ethanol precipitation in using NAoC and 100 % ethanol
I have tried to extract dna before and after CsCl step but the dna band are so dirty while phage genome extraction after cscl shows very faint genome band.
I need to extraction up to 5 ug of DNA. I will be so thankfull if somebody help me out.
respects and thanks
these links are useful
https://harmslab.uoregon.edu/.../preparation-of-phage-illumina-seque..
https://www.researchgate.net/.../Cost-effective_way_for_phage_DNA...
Article Preparation of a Phage DNA Fragment Library for Whole Genome...
regards
How large is the initial liquid culture, and what fraction of the final prep did you check on the gel?
Dear Alejandro thanks for the answer. Initial liquid was around 2L and the final prep was 2ul of he eluted DNA.
So, you have 2000 mL of a 10E8 pfu/mL suspension of a 20kb phage. Assuming 1 pfu = 1 virion, you'd have 2x10E11 genomes in total, which when divided by Avogadro's constant yields a total of 3.3x10E-13 moles (0.3 pmol). Assuming the 20 kb phage genome is double-stranded DNA, it would have an (average) molecular mass of 650x20,000 = 1.3x10E7 grams, so the 3.3x10E-13 moles represent approx. 4.3 ug in your culture. Assuming the purification has a 90% yield you should get a total of approx. 3.9 ug.
Those 2 uL you checked on the gel, what fraction do they represent of the total volume of purified DNA?
dear Alejandro, I really appreciate your wonderful answer,my fraction of total volume was 1 to 15th(2 ul of total elution of 30 ul) which in total I have concentration of 1ug....so I guess I need to increase my liquid culture up to 6L.
Mr aryan would you mind to tell me if you got a good protocol for phage DNA extraction
HI, I use PCI based extraction method followed by ethanol precipitation and 70% ethanol wash.
I make a phage titre up to 10^9 PFU/ml or more. In order to isolate phages out of liquid culture I treat them with PEG (10% w/v) and over night stirring at 4C .the mixture goes through centrifugation them at 7000 rpm for 1 hour followed by resuspension of the pellets in P.B.S.
phages are purified using ulteracenftrifuge and CSCl gradient.
Most important step is to make at least 1L of 10^9pfu/ml or higher of your bacteriophages.
We are using a modified method in which no PEG NACL is used and no CsCl is used. I can send u the protocol, if ur protocol is not working
The Phage Lysate is filtered by Syringe filter 0.22µm pore size ( Merck, Millipore), concentrated by centrifugal filters (Amicon Ultra-4, Ultracel-100K) to get maximum PFUs /ml and get rid of debris.then the following protocol
Genome extraction of the phage was performed using phenol Chloroform Isoamyl Alcohol (PCI) method, with some modifications. The phage lysate containing 10^11 bacteriophages, was filtered with a sterile syringe filter having pore size 0.22µm (Merck Millipore), DNase, RNase and 12.5µl of 1M MgCl2 were added in 1 ml of the filtrate and incubated at 37oC for 4 hours. The DNases and RNases were denatured at 75oC for 10 minutes in water bath and then shifted to 55oC and 50µl 0.5M EDTA, 40 µl 10%SDS and 2.5µl of proteinase K (20mg/ml) were added and incubated at 37oC for 1 hour while mixing gently after every 20 Mins. After 1 hour 550µl of the lysate is transferred to 2 Eppendorf tubes each and equal amount of PCI is added and mixed gently by inverting the tubes. The tubes are then centrifuged at 13000rpm for 10 Mins. The upper layer is picked carefully, and transferred to the new Eppendorf tubes and ice chilled 95% ethanol (1ml) and 50µl of ice chilled 3M sodium acetate is added and kept on ice for 5 Mins to facilitate precipitation, centrifuged at 13000rpm for 10 Mins. Pellet is formed, the supernatant is thrown away and 500µl of 70% ethanol is added in each tube and spun at 13000rpm for 5 Mins. The supernatant is thrown away and the tubes are inverted on blotting paper for drying. After 15-20Mins, DNase/RNase free water is added to dissolve DNA pellet. The tubes are kept overnight at room temperature for maximum dissolution. The genome thus obtained was run on 1% agarose gel in 1% TAE Buffer in the presence of a Ladder and visualized in a Gel documentation system.
Dear Iqbal Alvi, could you specify the concentration of DNAse and RNase you sude please?
is there any further treatment before sending the phage DNA for sequencing ?
As far as the DNA concentration and quality is well enough it is ready to go. No need for further treatment.
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