Sanger-based sequencing requires very specific DNA and primer concentrations for optimal performance. For template DNA like plasmids, DNA needs to be at a concentration of 50ng/ul in 10 ul. On top of that, a typical Sanger reaction requires 10 ul of primer at a concentration of 1 pMol/ul (note this is picomoles per microliter and NOT picomolar!)
A. You would like to send 5 DNA plasmids to be sequenced. You use a spectrophotomer to determine the concentrations of your samples (below). Fill out the table below as you would in your laboratory notebook to determine how much of each sample DNA and water you would have to mix to send these samples for sequencing.
|
Sample |
Concentration (ng/ul) |
For 50 ng/ul |
Water to 10 ul |
|
1 |
250.37 |
||
|
2 |
113.98 |
||
|
3 |
100.91 |
||
|
4 |
57.66 |
||
|
5 |
89.01 |
B. Now for the primer solution. A single sequencing PCR reaction requires 10 ul of primer solution at a concentration of 1 pMol/ul. Since you have 5 reactions, you will need 50 ul of primer solution. Your sequencing primer stock is at a concentration of 100 uM. How much primer and how much water would you have to mix to sequence all 5 reactions? Show your work.
