Lab 4: PROCEDURE
Restriction Digest
You will be digesting all three PCR products (the HCAII gene and the pETblue2 vector and the negative control) with DpnI. Fortunately for us, DpnI is functional in the PCR reaction buffer. If the enzyme was not compatible with this buffer, you would have to purify the DNA from the reaction, and then digest.
- Add 1 μL DpnI directly to each PCR tube. You should digest all three reactions – including the negative control
- Mix the reactions well by gently flicking the tube with your finger.
- Briefly centrifuge the samples and incubate at 37 °C for 1 hour.
DNA Purification Column
After the DpnI digests are complete, you will purify the DNA using small spin columns. Note you are also purifying your negative control. Use one column for each reaction. Be sure to label the spin columns so you don’t get them mixed up!
- Add 5× volume of PB buffer to each PCR reaction. (Example: if you have 100 μL of sample, add 500 μL PB). Mix well before moving on to step 3.
- Add the PB buffer + PCR reaction solution (from step 2), to the spin column and spin for 1 min. at 14,000 rpm (>16,000 rcf) in the centrifuge. Be sure to balance the tubes in the centrifuge before you start spinning. Your DNA should now be bound to the column; however, we often recommend that you save the flow-through at this point; if you do not end up with much DNA at the end of the purification, you can determine where you lost it (and whether it can still be recovered).
- Add 750 μL Buffer PE and spin for 1 min. at 14,000 rpm. Again, save the flow-through in the event that you lose your DNA and want to go back and find it later.
- Spin for 1 minute at 14,000 rpm again to dry the column. (Buffer PE contains ethanol, which will interfere with the Gibson assembly reactions if it is not completely removed).
- Place the column into a clean 1.5 mL eppendorf tube labeled with the name of the sample, the date, and your initials.
- Add 30 μL of Elution buffer (EB; 10 mM Tris pH 8.5) to the center of the column and incubate for 1 minute at room temperature.
- Spin for 1 minute at 14,000 rpm in the centrifuge. The flow-through contains your purified product. In order to get the maximum amount of DNA off of the column, add the flow-through back to the center of the column, incubate for one additional minute and then spin for 1 min. at 14,000 rpm in the centrifuge.
- SAVE THE FLOW-THROUGH CONTAINING YOUR DNA.
Determining the DNA Concentration
Once you have purified your PCR product, you will need to determine the concentration of your product. You will use the NanoDrop spectrophotometer to do this.
The following video demonstrates use of the NanoDrop:
- Place 2 μL of EB on the platform of the nanodrop and click on “BLANK” (upper left-hand corner) on the computer.
- To check your blank, place another 2 μL of EB on the platform of the NanoDrop and click on “MEASURE.” The curve should be close to flat and the DNA concentration should read approximately 0. If not, repeat st