How do i get rid of Formamide from my dsDNA?

mzahir89 · 10-08-2015, 02:30 AM

How do i get rid of Formamide from my dsDNA? Edit

I used hybridization buffer mix containing 70% formamide to renature my single stranded DNA. The hybridization was successfull as Qubit measured dsDNA. I now wish to get rid of formamide from my dsDNA since the viscosity does not allow me to proceed with shearing (Covaris) DNA for seq. Any suggestions?

Note the following methods did not work:

1. I have tried using precipitation method with isopropanol

2. I used both YM30 and YM50 purification columns to get rid of formamide

3. I used magnetic beads to pull out my DNA

4. I used a combination of 1/3 , 1/2, 2/3

5. I used 0.4 SSC containing Tween to wash away the formamide in 1:1 and 2:1 volumes following by 4 MQ washing steps - it only reduced the viscosity but did not get rid of it i.e. final product was still viscous.*

6. A combination of 1/5/2

Nothing above gave me my desired product.*

Appreciate any advise or feedback. Thanks.

nucacidhunter · 10-08-2015, 02:46 AM

Diluting dsDNA 70x and then a clean up using columns may be useful. Your dsDNA need to be large enugh not to be lost by passing through column.

mzahir89 · 10-08-2015, 02:51 AM

Yup, I have done that too! I can retain the DNA (similar starting and after purification DNA conc) but I still am not able to lose the formamide (which in my opinion is the cause of high viscosity).

nucacidhunter · 10-08-2015, 03:14 AM

I have not seen any evidence that would indicate binding formamide to silica columns. Diluting your dsDNA 70x will bring formamide concentration to 1% which is below tolerance of many silica based columns. There might be other reasons for viscousity of your DNA solution.

mzahir89 · 10-08-2015, 03:18 AM

This is the recipe of my hybridization mix:

• 50 ml dextran sulfate 20%
• 25ml deionized formamide
• 5ml 20x SSC
• 5ml 0.5M sodium phosphate buffer (pH = 6.5)
• 15ml milliQ Store at 4ºC, in dark

Any other ingredient in there that may be causing the viscosity?

nucacidhunter · 10-08-2015, 04:05 AM

Most likely cause of viscosity would be dextran sulfate. I hope someone with working experience of dextran can step in.
PS: You may end up doing phenol chloroform extraction.

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10-08-2015, 02:30 AM	#1
mzahir89 Junior Member Location: Groningen, The Netherlands Join Date: Aug 2015 Posts: 6	How do i get rid of Formamide from my dsDNA? How do i get rid of Formamide from my dsDNA? Edit I used hybridization buffer mix containing 70% formamide to renature my single stranded DNA. The hybridization was successfull as Qubit measured dsDNA. I now wish to get rid of formamide from my dsDNA since the viscosity does not allow me to proceed with shearing (Covaris) DNA for seq. Any suggestions? Note the following methods did not work: 1. I have tried using precipitation method with isopropanol 2. I used both YM30 and YM50 purification columns to get rid of formamide 3. I used magnetic beads to pull out my DNA 4. I used a combination of 1/3 , 1/2, 2/3 5. I used 0.4 SSC containing Tween to wash away the formamide in 1:1 and 2:1 volumes following by 4 MQ washing steps - it only reduced the viscosity but did not get rid of it i.e. final product was still viscous.* 6. A combination of 1/5/2 Nothing above gave me my desired product.* Appreciate any advise or feedback. Thanks.

10-08-2015, 04:05 AM	#6
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Most likely cause of viscosity would be dextran sulfate. I hope someone with working experience of dextran can step in. PS: You may end up doing phenol chloroform extraction. Last edited by nucacidhunter; 10-08-2015 at 05:14 AM.

10-08-2015, 02:46 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Diluting dsDNA 70x and then a clean up using columns may be useful. Your dsDNA need to be large enugh not to be lost by passing through column.

10-08-2015, 02:51 AM	#3
mzahir89 Junior Member Location: Groningen, The Netherlands Join Date: Aug 2015 Posts: 6	Yup, I have done that too! I can retain the DNA (similar starting and after purification DNA conc) but I still am not able to lose the formamide (which in my opinion is the cause of high viscosity).

10-08-2015, 03:14 AM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	I have not seen any evidence that would indicate binding formamide to silica columns. Diluting your dsDNA 70x will bring formamide concentration to 1% which is below tolerance of many silica based columns. There might be other reasons for viscousity of your DNA solution.

10-08-2015, 03:18 AM	#5
mzahir89 Junior Member Location: Groningen, The Netherlands Join Date: Aug 2015 Posts: 6	This is the recipe of my hybridization mix: • 50 ml dextran sulfate 20% • 25ml deionized formamide • 5ml 20x SSC • 5ml 0.5M sodium phosphate buffer (pH = 6.5) • 15ml milliQ Store at 4ºC, in dark Any other ingredient in there that may be causing the viscosity?