McClean: Anneal and Extend: Difference between revisions
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==Protocol== | ==Protocol== | ||
# Design oligos so that they have ~20bp of overlap. | |||
# Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O. | # Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O. | ||
# For one reaction the PCR mix should be: | # For one reaction the PCR mix should be: | ||
Line 19: | Line 19: | ||
#*33.5μL H<sub>2</sub>O | #*33.5μL H<sub>2</sub>O | ||
#Run in a thermocycler as follows (notice that we do not use many cycles): | #Run in a thermocycler as follows (notice that we do not use many cycles): | ||
#*94°C for 5 min | |||
**98°C for 10s | #*Repeat the following three lines 2-3 times: | ||
**53°C for 20s | #**98°C for 10s | ||
**72°C for 30s | #**53°C for 20s | ||
#**72°C for 30s | |||
#*72°C 5 min | |||
==Notes== | ==Notes== | ||
<!-- 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. | |||
==References== | ==References== | ||
==Contact== | ==Contact== | ||
<!--Change the information below to your info if you add a new protocol--> | <!--Change the information below to your info if you add a new protocol--> | ||
*'''[[User:Megan N McClean|Megan N McClean]] 14:01, | *'''[[User:Megan N McClean|Megan N McClean]] 14:01, 19 September 2012 (EDT)''' | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. |
Latest revision as of 14:48, 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
- Design oligos so that they have ~20bp of overlap.
- 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
Contact
- Megan N McClean 14:01, 19 September 2012 (EDT)
or instead, discuss this protocol.