<commit_before># Quantify Extracted DNA Using High-throughput Quant-iT dsDNA Assay Kit (High Sensitivity)   
Author: <NAME>

## Objectives:   
Accurately quantify the amount of total DNA extracted using 96 well plate format fluorescent assays.   

## Materials:  
1. Invitrogen Quant-iT dsDNA high senstiviely assay kit (Thermo Scientific: Q33120)   
2. Black 96-well plates (VWR: 62403-156)   
3. Fluorescent microtiter plate reader (contact: <NAME> <<EMAIL>> over in Biorenewable building)   
4. 50ml sterile tubes for reagent mixing   
5. Pipettemen and tips   

## Procedures:   
### Preparation    
#### Days before you do the quantification:    
-[ ] 1. Make sure you have enough 96-well black plates.  
         + One column of each plate will be used for DNA concentration standards.   
         + If you have 1 96-well full of DNA samples, you will need to use 2 96-well plates for quantification.   
-[ ] 2. Decide your quantification plate layout.  
         + ie., which column of your DNA samples would be moved to plate 2 to be quantified.   
-[ ] 3. Contact <NAME> <<EMAIL>> to make sure that we can use their fluorescent plate reader on a particular day and time.   
         + The reading only takes ~ 5 min per plate. But the machine is shared by many labs. Make sure we don't introduce conflicts.   
         + They have both visible light plate reader and fluorescent plate reader. Make sure you are using the fluorescent one.   

#### 30 min - 1 hr before you do the quantification:   
-[ ] 1. Bring the **Quant-iT dsDNA HS buffer (Component B)** to room temperature.   
         + Located in the fridge in the lab (in the Quant-iT dsDNA HS kit box)   
         + Protect from the light (put it in one of the drawers)   
-[ ] 2. Locate the **Quant-iT dsDNA HS reagent (Component A)**.   
         + The fluorescent dye will freeze at 4ºC. Therefore, it's kept at room temperature to avoid freeze and thaw.   
         + In the **Qubit dsDNA HS Assay Kit** box in the drawer labeled **DNA quantification** under the Qubit.   
         + There are two fluorescent dyes. Make sure take the one says **for 96 well** on it.   
         + Put it in the same drawer where you put the buffer in **Step 1**, also protect from light.   
-[ ] 3. Thaw your DNA samples.  
         + Give it a quick vortex to mix.  
         + Spin all of the liquid to the bottom of the plate using the plate spinner.   

### Detailed steps:   
-[ ] 1. Prepare the working solution in a 50ml sterile tube.   
         + For each sample, working solution consists buffer:dye = 200:1 (ie., 199µl buffer + 1µl dye)   
         + Because we are using medium reservoir and multichannel pipettmen, make sure you mix some extra.  
             + I usually prpare the working solution for 100 samples for 1 96 well plate.   
             + ie., for one plate, I will need:   
                 + 100 X 199µl buffer = 19ml and 900µl of buffer    
                 + 100 X 1µl dye = 100µl of dye   
-[ ] 2. Bring the DNA standards out of the fridge.  
         + In the Quant-iT dsDNA HS kit box (8 screw cap tubes of them)   
-[ ] 3. Read the label to arrange them in order      
-[ ] 4. Tap each tube to mix    
-[ ] 5. Do a quick spin to make sure all of the liquid falls to the bottom of each tube   
         + This also helps to prevent contamination    
-[ ] 6. Arrange the standards in order in a tube holder.  
         + Put the tubes next to each other leave without space.   
         + Arrange them from 0 - 10ng/µl    
-[ ] 7. Label a black 96-well plate **plate 1**.  
-[ ] 8. Pour the working solution from **Step 1** into a medium reservoir.   
-[ ] 9. Use the E1-cliptip 8 channel multipipetteman add 200µl of working solution into each well.  
         + Use program `repeater_200µl`    
         + Use 8 1250µl cliptips      
-[ ] 10. Refer to your layout, to the column designed to standards, add 10µl of each DNA standard.  
         + Uncap your DNA standard tubes  
         + Used the Adjustable Spacing E1-Cliptip 8 Channel multipepetteman   
         + Use 8 1250µl cliptips  
-[ ] 11. Lay a piece of Kim-wipe on the bench top.  
-[ ] 12. Carefully take off your plate sealer. Lay it upside down on the Kim-wipe you just laid down.  
-[ ] 13. Use a VWR manual 10µl 8 channel pipetteman, add 2µl of your DNA samples to the designated wells in **Plate 1**.  
         + Pipette up and down gently to mix.   

<commit_msg>finished quantit dsdna sop<commit_after># Quantify Extracted DNA Using High-throughput Quant-iT dsDNA Assay Kit (High Sensitivity)   
Author: <NAME>

## Objectives:   
Accurately quantify the amount of total DNA extracted using 96 well plate format fluorescent assays.   

## Materials:  
1. Invitrogen Quant-iT dsDNA high senstiviely assay kit (Thermo Scientific: Q33120)   
2. Black 96-well plates (VWR: 62403-156)   
3. Fluorescent microtiter plate reader (contact: <NAME> <<EMAIL>> over in Biorenewable building)   
4. 50ml sterile tubes for reagent mixing   
5. E1-cliptip 8-channel adjustable spacing pipetteman   
6. VWR manual 8-channel pipetteman 10µl   
7. Autoclaved cliptips (less than 1 box)    
8. VWR 10XL autoclaved tips:   
    + N = number of plates of samples   
    + the number of boxes of 10XL tips needed: N+1        

## Procedures:   
### Preparation    
#### Days before you do the quantification:    
-[ ] 1. Make sure you have enough 96-well black plates.  
         + One column of each plate will be used for DNA concentration standards.   
         + If you have 1 96-well full of DNA samples, you will need to use 2 96-well plates for quantification.   
-[ ] 2. Make sure you have enough of 10XL VWR tips.   
-[ ] 3. Decide your quantification plate layout.  
         + ie., which column of your DNA samples would be moved to plate 2 to be quantified.   
-[ ] 4. Contact <NAME> <<EMAIL>> to make sure that we can use their fluorescent plate reader on a particular day and time.   
         + The reading only takes ~ 5 min per plate. But the machine is shared by many labs. Make sure we don't introduce conflicts.   
         + They have both visible light plate reader and fluorescent plate reader. Make sure you are using the fluorescent one.   

#### 30 min - 1 hr before you do the quantification:   
-[ ] 1. Bring the **Quant-iT dsDNA HS buffer (Component B)** to room temperature.   
         + Located in the fridge in the lab (in the Quant-iT dsDNA HS kit box)   
         + Protect from the light (put it in one of the drawers)   
-[ ] 2. Locate the **Quant-iT dsDNA HS reagent (Component A)**.   
         + The fluorescent dye will freeze at 4ºC. Therefore, it's kept at room temperature to avoid freeze and thaw.   
         + In the **Qubit dsDNA HS Assay Kit** box in the drawer labeled **DNA quantification** under the Qubit.   
         + There are two fluorescent dyes. Make sure take the one says **for 96 well** on it.   
         + Put it in the same drawer where you put the buffer in **Step 1**, also protect from light.   
-[ ] 3. Thaw your DNA samples.  
         + Once it's thaw, give it a quick vortex to mix.  
         + Spin all of the liquid to the bottom of the plate using the plate spinner.   

### Detailed steps:   
**NOTE:**    
**The final volume of each well will be different. For all sample wells, each well will end up with 202µl. For standard wells, each well will end up with 210µl. I called Thermo-Fisher, they said that the assay is designed to be this way (instead of equal volume). They also said that their results show no difference between different volumes and equal volumes.** 

-[ ] 1. Wipe the bench top clean with 70% ethanol (EtOH).   
-[ ] 2. Prepare the working solution in a 50ml sterile tube.   
         + For each sample, working solution consists buffer:dye = 200:1 (ie., 199µl buffer + 1µl dye)   
         + Because we are using medium reservoir and multichannel pipettmen, make sure you mix some extra.  
             + I usually prpare the working solution for 100 samples for 1 96 well plate.   
             + ie., for one plate, I will need:   
                 + 100 X 199µl buffer = 19ml and 900µl of buffer    
                 + 100 X 1µl dye = 100µl of dye   
-[ ] 3. Bring the DNA standards out of the fridge.  
         + In the Quant-iT dsDNA HS kit box (8 screw cap tubes of them)   
-[ ] 4. Read the label to arrange them in order      
-[ ] 5. Tap each tube to mix    
-[ ] 6. Do a quick spin to make sure all of the liquid falls to the bottom of each tube   
         + This also helps to prevent contamination    
-[ ] 7. Arrange the standards in order in a tube holder.  
         + Put the tubes next to each other leave without space.   
         + Arrange them from 0 - 10ng/µl    
-[ ] 8. Take a black plate. Put the lid aside if there is one.   
-[ ] 9. Label the side body of the black 96-well plate **plate 1**.   
         + Use a silver Sharpie     
-[ ] 10. Pour the working solution from **Step 1** into a medium reservoir.   
-[ ] 11. Use the E1-cliptip 8 channel multipipetteman add 200µl of working solution into each well.  
         + Use program `repeater_200µl`    
         + Use 8 1250µl cliptips      
-[ ] 12. Refer to your layout, to the column designed to standards, add 10µl of each DNA standard.  
         + Uncap your DNA standard tubes  
         + Used the Adjustable Spacing E1-Cliptip 8 Channel multipepetteman   
         + Use 8 1250µl cliptips  
-[ ] 13. Lay a piece of Kim-wipe on the bench top.  
-[ ] 14. Carefully take off your plate sealer. Lay it upside down on the Kim-wipe you just laid down.  
-[ ] 15. Use a VWR manual 8 channel pipetteman (10µl), add 2µl of your DNA samples to the designated wells in **Plate 1**.  
         + Pipette up and down gently to mix.   
         + Depending on your samples, you may need to adjust the volume of your samples.
         + In geeral, 1µl tends to go below detection and 3µl tends to go over detection limits.   
         + Anyway, I find 2µl works very well most of the time. But you still may need to redo some samples afterwards.  
-[ ] 16. Put the black plate lid back on if there is one. Put the plate in an empty drawer to protect it from light.  
-[ ] 17. Put your sample DNA plate back into the -80ºC freezer.    
-[ ] 18. Repeat **Step 8** to **Step 17** for more plates of samples.   
-[ ] 19. Once all samples are done, stack them together and cover with foil.  
-[ ] 20. Bring a flash drive. Carry over to the fluorescent plate reader.   
         + Be careful. Don't spill!   
-[ ] 21. Do the end-point reading using an existing template.   
         + Protocol fluorescent settings:    
             + excitation maxima: ~502 nm
             + emission maxima: ~523 nm    
         + Settings on the fluorescent plate reader we are using:  
             + excitation: 485/20 nm    
             + emission: 516/20 nm  
-[ ] 22. Export the readings and save it to your flash drive.   
-[ ] 23. Clean up:  
         -[ ] Put the **Quant-iT buffer (component B)** back into the fridge.   
         -[ ] Put the **Quant-iT reagent (component A)** back into the labeled foil and into the drawer under Qubit.   
         -[ ] Throw away the black plates.   
         -[ ] Wipe the bench top clean with 70% ethanol.   
         -[ ] Wipe the pipettemen clean with 70% ethanol.   
         


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