Aug 06, 2022
Annealing Oligonucleotides (Instructor Protocol)
This protocol is a draft, published without a DOI.
- 1University of Wisconsin - Stout
- Yeast ORFans CURE
Protocol Citation: Brian P P Teague 2022. Annealing Oligonucleotides (Instructor Protocol). protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 27, 2022
Last Modified: August 06, 2022
Protocol Integer ID: 67670
Keywords: annealing oligonucleotide, instructor protocol, protocol
Abstract
Image Attribution
By Smokefoot - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=117155213
Materials
- TE Buffer
Note
We resuspend and dilute oligonucleotides in TE, not water. The Tris keeps the pH at 8.0, which is good for stability, and the EDTA chelates ions and prevents nuclease degredation.
Note
I do not trust my ability to prepare nuclease-free TE buffer, so we usually order it from a manufacturer (it's not expensive.) However, if you do prepare your own, make sure to autoclave it to destroy DNAses!
- 15 ml conical tubes
Equipment:
- Microfuge with PCR tube rotor
- Thermocycler
Protocol materials
15 ml conical tubes
TE Buffer
Safety warnings
TE is not hazardous; neither are synthetic DNA oligonucleotides.
HOWEVER, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats and gloves to keep your samples nuclease-free.
Lab Setup
Prepare 15 ml conicals with 5 mL TE Buffer in each. Prepare one tube for each 4 students (2 groups of 2).
Program a thermocycler with the following program:
- Step 1: 95°C for two minutes
- Step 2: 95°C for 45 seconds, decreasing one degree each cycle
- Step 3: Go to step 2 70 times
- Step 4: Store at 8°C
Instructor Tips & Common Student Errors
Instructor Tips
- This is the first "real" lab. I generally start the class explaining what annealing is and why we're doing it, point out where the materials are, and then sit down. My approach is deliberately hands-off.
- Each semester, I am amazed that this protocol takes students an entire two-hour lab period. It probably has to do with my hands-off approach, as mentioned above.
- Students often struggle with the dilution math. Remind them of c1 * v1 = c2 * v2 and remind them that they've already done this once. Suggest that they review the dilutions and pipetting lab and that they ask their colleagues for help. Only if students are really stuck will I review the procedure with them.
- We've only got one thermocycler -- but this reaction is fine to wait until the entire class has loaded their samples to start. (As opposed to the PCR, which needs to stay on ice until it's time to start.) So I ask students to load their samples into the thermocycler as they finish.
- As students load their samples, double-check that they look like about 10 µl of liquid. Significantly more may be a sign of a pipetting error.
- As students load their samples, double-check that they're labeled. Remind them that their tube will be one of many and ask if they'll be able to pick their tube out from all the others?
- Remind students to record their tube label in their notebook.
Common Student Errors
- Difficulty doing the dilution math.
- Didn't mix the sample well
- Didn't label their tubes