Griffitts:PCR primers: Difference between revisions
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==Primer Design== | ==Primer Design== | ||
===General=== | ===General=== | ||
* Primers should generally be 20 | * Primers should generally be 20–22 nt long with 10–12 Gs and Cs | ||
* Remember to use the reverse compliment for your reverse primer | * Remember to use the reverse compliment for your reverse primer | ||
===Sequencing=== | ===Sequencing=== | ||
| Line 19: | Line 21: | ||
* Find the nmoles on the tube (e.g. 24.65 nmoles) | * Find the nmoles on the tube (e.g. 24.65 nmoles) | ||
* Multiply by ten (e.g. 24.65 × 10 = 246.5) | * Multiply by ten (e.g. 24.65 × 10 = 246.5) | ||
* Take that number and add that many μL of [[Griffitts: | * Take that number and add that many μL of [[Griffitts:Common buffers#TE buffer|TE buffer]] (e.g. add 240 to 250 μL) | ||
* This will give you | * This will give you 100× primers (i.e. 100 µM) | ||
Latest revision as of 08:23, 19 April 2013
Primer Design
General
- Primers should generally be 20–22 nt long with 10–12 Gs and Cs
- Remember to use the reverse compliment for your reverse primer
Sequencing
- Design one forward primer for every ~500 bp
- You won't get good sequence where your primer sits, so design your first primer to be a little upstream from your sequence
- You only need one reverse primer
Cloning
- Check for restriction sites within your target before adding them to your primers
- Add a "CGC" to the ends of your primers
Gene Deletion
- Check for restriction sites within your target before adding them to your primers
- Add a "CGC" to the ends of your primers
- Clone ~500 bp upstream and downstream of the gene you're intending to delete and include ~20 bp of the target gene
- For your internal primers, give them 10-12 bp homology with each other for Overlap-extension PCR
Primer Hydration
- Find the nmoles on the tube (e.g. 24.65 nmoles)
- Multiply by ten (e.g. 24.65 × 10 = 246.5)
- Take that number and add that many μL of TE buffer (e.g. add 240 to 250 μL)
- This will give you 100× primers (i.e. 100 µM)
