Could anyone help me find the best composition for cell lysis solution for manual DNA extraction using blood as the source? I want to prepare the solution in house. Does anyone have experience preparing it?
- See reference to a previous answer and attached
- https://www.researchgate.net/post/Best_cell_lysis_solution_for_DNA_extraction_by_manual_method?isAnswerFieldFocused=true
- In essence, you require a sligtly hypertonic buffered solution to burst cells and with EDTA to make things stable:
- This can be the buffer in attached doc - which in fact is specifically used for the purpose of red blood cell DNA extraction
- Alternatively you can use TNE buffer:
- TNE Buffer 50 mm Tris–HCl (pH 7.4) 100 mm NaCl 0.1 mm EDTA
- Plus or minus 0.1% SDS
- Having lysed your cells in TNE +/- SDS or in the salt buffer described you then incubate with Proteinase K to digest away protein (55C 1 hr or 37C over night)
- It is this deproteinated lysate that you then subjecty o purification; either by Trizol or phenol chloroform extraction or by passing down a commercial column
- i have used all such methods for a plethora of sample types - including blood - and they all work well and are underpinned by the same purification regimen
You can use different detergent like CHAPS, SDS, Tween-80 or NP-40. But its is very important to separate the protein from the DNA in order to get pure DNA which can be further used for the PCR, Blotting or various purposes. You can use Proteinase K for protein separation.
You can use Chelex-100 for the extraction of genomic DNA. It is economical and serves to perform PCR.
The description is on this page.
https://www.dovepress.com/methods-for-extracting-genomic-dna-from-whole-blood-samples-current-pe-peer-reviewed-fulltext-article-BSAM
Best regards,
DD-D
- Chelex are excellent
- I have used too
- But best to Mechanically lyse into one of the buffers described
Hi Sam vijay kumar .J
I am using following protocol for a long time. It is really useful.
- 10 mM Tris-HCI
- 320 mM Sucrose (adding sucrose after autoclave is a way better.)
- 5 mM MgCI2
- and distilled water.
- Adjust the pH to 8.0 with 40% NaOH .
- Autoclave and add triton X-100 (10 ml for 1 L buffer). Store at 4o C.
Add this buffer onto your blood sample. ( You can use 20 ml for 1-2 ml blood sample.)
Shake at RT for a while (4 min is enough) and centrifuge for 10 min at 3000 rpm.
Discard supernatant and resuspend the cell pellet in 10 ml of this buffer. Shake. Centrifuge. (same parameters). Discard supernatant.
2. Then you can lyse your cells
Prepare a buffer containing
- 400 mM Tris-HCL
- 0.5 M EDTA
- 150 mM NaCI
- and distilled water.
- Adjust the pH to 8.0 with 40% NaOH.
- Autoclave and add 1% SDS (You can store this at RT.)
- Add 1 ml of this buffer to your cell pellet. Vortex. Resuspend the pellet.
- Add sodium perchlorate (250-500 ul) and shake (app. 10 min. at RT)
- Incubate for 20-25 min at 65 oC and quickly vortex every 5 min.
Then, you will be able to extract your DNA.
Best,
Elmas
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