TITLE: dsDNA quantification using Sybr Green I
AUTHORS: James JN Kitson

[DESCRIPTION]
This is a simple protocol that uses Sybr Green 1 and a microplate reader to quantify dsDNA concentrations in unknown samples. This is best suited for situations where a high number of samples need to be quantified (e.g. normalisation of samples before pooling for Illumina sequencing). For smaller sample numbers (e.g. < 50 samples) it will be more efficient to simply use a qubit or nanodrop.

You need to pick an appropriate standard concentration range for the type of samples you will be testing. For genomic DNA it is probably better to have a lower concentration range (0-10 ng/μl) whereas for PCR products, standards will probably need to be much more concentrated (0-50 or maybe even 100ng/μl).

[STEPS]
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
1. Select which standards we need to make and use based on the strength of the samples being tested.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
3.  Serially dilute this as below:
          			 				Dilution 			 			 				lambda 				DNA (µL) 			 			 				TE 				(µL) 			 			 				[lambda 				DNA] (ng/μl) 			 		  			 				1 			 			 				1000 				(from 10 µg/mL stock) 			 			 				0 			 			 				10 			 		  			 				2 			 			 				500 				(from dilution 1) 			 			 				500 			 			 				5 			 		  			 				3 			 			 				500 				(from dilution 2) 			 			 				500 			 			 				2.5 			 		  			 				4 			 			 				500 				(from dilution 3) 			 			 				500 			 			 				1.25 			 		  			 				5 			 			 				500 				(from dilution 4) 			 			 				500 			 			 				0.625 			 		  			 				6 			 			 				500 				(from dilution 5) 			 			 				500 			 			 				0.313 			 		  			 				7 			 			 				500 				(from dilution 6) 			 			 				500 			 			 				0.156 			 		  			 				8 			 			 				0 				(from dilution 7) 			 			 				500 			 			 				0.000 			 		 
 
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
3. Aliquot 25 μl of each of these into PCR strips and store at -20 °c until needed. Thaw one for each run on the plate reader.   
 If you have previously made your dilution series start at this point.
SECTION: Part 2 - Make and distribute Sybr Green 1 working solution.
4. Make a 100x Sybr Green 1 solution (5ul Sybr Green 1+ 495 μl of TE).
This can be pipetted out into 50 - 100 μl aliquots and stored at -20 °c. Once thawed, do not reuse.
96 μl of 100x Sybr Green 1 is required per plate of samples.
 If you have previously made your 100X Sybr Green 1 start at this point.
SECTION: Part 2 - Make and distribute Sybr Green 1 working solution.
5. Make a solution of 1x Syber Green 1 as below:
 

SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
6. Distribute 95 μl of Sybr Green 1 working solution into the first 4 columns of a Nunc black microtiter plate and 100 μl into the last 8 columns.
 This is best done using a multichannel electronic repeater or 96 well pipette if you are testing a lot of samples.
SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
7. Using a p10 multichannel pipette, add 5ul of each standard into the first 4 columns of the control plate as shown in figure 1.
  

SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
8. Distribute 98 μl of sybr green 1 working solution into n wells of a nunc black microtiter plate. 
Use multiple plates for more than 96 samples.
SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
9. Add 2 μl of sample dsDNA into each of the sample wells.
SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
10. Seal with a clear plate seal.
SECTION: Part 3 - dilute Sybr Green 1 working solution and add DNA.
11. Incubate at room temperature in the dark for 10 minutes.   10 min 

SECTION: Part 4 - load microplate reader and run the assay.
12. Run samples on your plate reader according to manufacturers instructions with an excitation wavelength of 485 nM and an emission wavelength of 535 nM.


SECTION: Part 4 - load microplate reader and run the assay.
13. Check that your calibration curve has a very high R2 value (greater than 0.95) and multiply your measured concentrations by 2.5 to get a final concentration.

  

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
2. Using NEB lambda DNA (item N3011S) make a 100 ng/μl dsDNA standard in TE (100 μl of 500ng/μl lambda DNA + 400 μl of TE).
TE =10 mM Tris HCI, 1 mM EDTA, pH 8
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
3.  Serially dilute this as below:
          			 				Dilution 			 			 				lambda 				DNA (µL) 			 			 				TE 				(µL) 			 			 				[lambda 				DNA] (ng/μl) 			 		  			 				1 			 			 				1000 				(from 10 µg/mL stock) 			 			 				0 			 			 				10 			 		  			 				2 			 			 				500 				(from dilution 1) 			 			 				500 			 			 				5 			 		  			 				3 			 			 				500 				(from dilution 2) 			 			 				500 			 			 				2.5 			 		  			 				4 			 			 				500 				(from dilution 3) 			 			 				500 			 			 				1.25 			 		  			 				5 			 			 				500 				(from dilution 4) 			 			 				500 			 			 				0.625 			 		  			 				6 			 			 				500 				(from dilution 5) 			 			 				500 			 			 				0.313 			 		  			 				7 			 			 				500 				(from dilution 6) 			 			 				500 			 			 				0.156 			 		  			 				8 			 			 				0 				(from dilution 7) 			 			 				500 			 			 				0.000 			 		 
 
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
4. Aliquot 20 μl of each of these into PCR strips and store at -20 °c until needed. Thaw one for each run on the plate reader.   
 If you have previously made your dilution series start at this point.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
5. Make a 100x Sybr Green 1 solution (5ul Sybr Green 1+ 495 μl of TE).
This can be pipetted out into 50 - 100 μl aliquots and stored at -20 °c. Once thawed, do not reuse.
96 μl of 100x Sybr Green 1 is required per plate of samples.
 If you have previously made your 100X Sybr Green 1 start at this point.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
6. Make a solution of 1x Syber Green 1 as below:
 

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
7. Distribute 98 μl of Sybr Green 1 working solution into the first 4 columns of a Nunc black microtiter plate and 100 μl into the last 8 columns.
 This is best done using a multichannel electronic repeater or 96 well pipette if you are testing a lot of samples.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
9. Distribute 98 μl of sybr green 1 working solution into n wells of a nunc black microtiter plate. 
Use multiple plates for more than 96 samples.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
10. Add 2 μl of sample dsDNA into each of the sample wells.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
11. Seal with a clear plate seal.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
12. Incubate at room temperature in the dark for 10 minutes.   10 min 

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
13. Run samples on your plate reader according to manufacturers instructions with an excitation wavelength of 485 nM and an emission wavelength of 535 nM.


SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
14. Check that your calibration curve has a very high R2 value (greater than 0.95) and multiply your measured concentrations by 2.5 to get a final concentration.

  

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
2. Using NEB lambda DNA (item N3011S) make a 10 ng/μl dsDNA standard in TE (20ul of 500ng/μl lambda DNA + 980 μl of TE).
TE =10 mM Tris HCI, 1 mM EDTA, pH 8
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
4. Aliquot 20 μl of each of these into PCR strips and store at -20 °c until needed. Thaw one for each run on the plate reader.   
 If you have previously made your dilution series start at this point.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
5. Make a 100x Sybr Green 1 solution (5ul Sybr Green 1+ 495 μl of TE).
This can be pipetted out into 50 - 100 μl aliquots and stored at -20 °c. Once thawed, do not reuse.
96 μl of 100x Sybr Green 1 is required per plate of samples.
 If you have previously made your 100X Sybr Green 1 start at this point.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
6. Make a solution of 1x Syber Green 1 as below:
 

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
7. Distribute 98 μl of Sybr Green 1 working solution into the first 4 columns of a Nunc black microtiter plate and 100 μl into the last 8 columns.
 This is best done using a multichannel electronic repeater or 96 well pipette if you are testing a lot of samples.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
8. Using a p10 multichannel pipette, add 2ul of each standard into the first 4 columns of the control plate as shown in figure 1.
   
 


SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
9. Distribute 98 μl of sybr green 1 working solution into n wells of a nunc black microtiter plate. 
Use multiple plates for more than 96 samples.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
10. Add 2 μl of sample dsDNA into each of the sample wells.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
11. Seal with a clear plate seal.
SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
12. Incubate at room temperature in the dark for 10 minutes.   10 min 

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
13. Run samples on your plate reader according to manufacturers instructions with an excitation wavelength of 485 nM and an emission wavelength of 535 nM.


SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
14. Check that your calibration curve has a very high R2 value (greater than 0.95) and multiply your measured concentrations by 2.5 to get a final concentration.

  

SECTION: Part 1 - Make a dilution series of a Lambda DNA standard:
8. Using a p10 multichannel pipette, add 2ul of each standard into the first 4 columns of the control plate as shown in figure 1.