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
#*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 5 min
#**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]]''' When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit.  It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.
*[[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]]''' Different tetrad dissection protocols call for using different enzymes to digest the ascus wall.  Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail).  Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product.  β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).


==References==
==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==
==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, 20 July 2011 (EDT)'''
*'''[[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

  1. Design oligos so that they have ~20bp of overlap.
  2. Dilute oligos to 25μM from the -80°C stock. Dilute into H2O.
  3. 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
  4. 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

or instead, discuss this protocol.


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