Sequencing

After you finish purifying your PCR product, measure quality and quantity with NanoDrop. To send samples out for sequencing to GeneWiz the concentration of purified PCR product needs to be around 5 ng/ul.

Diluting Purified PCR Product
Use C1V1 = C2V2

• C1 = Concentration of DNA measured (ng/ul)
• V1 = The total amount of elution buffer (EB) you used
• C2 = 5 ng/ul this is the concentration we want
• V2 = The total volume we will have after diluting

You will be solving for V2 = Final volume
To find out how much molecular grade water we need to add we subtract the amount of EB we used from Final volume.

Purification Kit Tip
Whatever method you used to setup your PCR (Genscript or Super mix) it is sometimes hard to recall the amount sitting in your tubes after you have run your gel especially if it has been a few days since you ran your gel. If you used the Super mix then your PCR reaction was 25 ul and then you loaded 5 ul into the gel to see if your PCR was successful leaving you with 20 ul (45 ul if you used Genscript).

Using the Qiagen PCR Purification kit (cat no. ###) the first step ask you to add 5 parts of PB solution to 1 part PCR product. This means you are multiplying the amount in your PCR tube 20 ul (or 45 ul) by 5. The rest of the protocol is easy to follow.

Quick Reference 1:5 Primer dilution

• Vortex primer 100x master stock for 30 sec
• 95 ul 1x TAE
• 5 ul of 100x Master stock
• Vortex working stock for 30 sec before using

Alternate approach
Primer concentration for sequencing is half the concentration used for PCR. You can keep it really simple and take a few microliters from the primers used for PCR and an equal amount of molecular grade water or 1x TAE buffer.

This is how to calculate the 1:5 dilution (so you can make what ever volume you want)
Use C1V1 = C2V2 (again)

• C1 = 100x Master stock
• V1 = How many microliters of 100x master stock to transfer
• C2 = 5x concentration we want
• V2 = 100 ul (total volume we will end up having)

You will solve for V1. No additional steps required.

Quick Reference

• 10 ul of of template
• 5 ul of diluted primer

Steps
After purifying your PCR product, you will need to measure the concentration with NanoDrop. Optimal concentration is around 5 ng/ul. More than likely you will have to dilute the samples.

Tubes or Plates
If you are sending 48 samples or less, use the 8-strip 0.2 ml tubes with caps and label the side of the tube with initials and sample number. Very important: You have to use the initials the account holder. As of now the only account hold is the lab manager.
K    K
K    K
0    0
1     2

If you have more than 48 samples to send out, use a 96 well plate.

Loading Samples
To each tube (or well) you will add 10 ul of your template and 5 ul of diluted primer

Important to remember you will only put one primer per tube. ie: Tube 01 will have forward primer and tube 02 will have reverse primer – both will have the same template.