McClean: Anneal and Extend: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Notes) |
(→Notes) |
||
Line 29: | Line 29: | ||
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | <!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | ||
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | ||
*[[User:Megan N McClean|Megan N McClean]]It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well. | *[[User:Megan N McClean|Megan N McClean]] It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well. | ||
==References== | ==References== |
Revision as of 14:47, 19 September 2012
Overview
This protocol describes a simple procedure to annealing and extending two oligos (with homology) to get double-stranded DNA.
Materials
- Oligos (25μM; take the standard McClean Lab -80°C stock which is at 100μM in TE and dilute it 1:4 in H2O)
- Takara PCR reagents (see: Takara PrimeStar PCR)
Protocol
- Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
- For one reaction the PCR mix should be:
- 10μL 5X Takara Buffer
- 4μL 2.5mM DNTPs
- 0.5μL Takara polymerase
- 1 μL 25μM forward oligo
- 1 μL 25μM reverse oligo
- 33.5μL H2O
- Run in a thermocycler as follows (notice that we do not use many cycles):
- 94°C for 5 min
- Repeat the following three lines 2-3 times:
- 98°C for 10s
- 53°C for 20s
- 72°C for 30s
- 72°C 5 min
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Megan N McClean It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.
References
Adapted from Maitreya Dunham's protocol (http://dunham.gs.washington.edu/sporulationdissection.htm) and the Botstein lab protocol (http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf)
Contact
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.