McClean:Colony PCR(E. coli)

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'''*[[User:Megan N McClean|Megan N McClean]] 15:07, 29 April 2012 (EDT)''': We really don't need to be doing 50μL reactions to check colonies.  If someone has the time, please try a smaller volume (20μL) of this protocol and see if it works and then update this protocol with a note.
'''*[[User:Megan N McClean|Megan N McClean]] 15:07, 29 April 2012 (EDT)''': We really don't need to be doing 50μL reactions to check colonies.  If someone has the time, please try a smaller volume (20μL) of this protocol and see if it works and then update this protocol with a note.
 +
 +
*'''[[User:Michael T. Patel|Michael T. Patel]] 17:33, 26 April 2013 (EDT)''': Running reaction volumes of 25μL works fine, though you should use larger master mixtures and allotting at the end to avoid pipetting error with small volumes. Also you can probably use reaction volumes smaller than 25μL, though you may want to consider adding mineral oil to these.
==References==
==References==

Revision as of 17:33, 26 April 2013


Contents

Overview

Protocol for e. coli colony PCR. Useful for checking if you've got the desired plasmid after you've transformed a ligation, or similar.


Materials

  • Sterile toothpicks or loops.
  • HotMaster Taq Polymerase
  • 10x HotMaster Buffer with Mg2+
    • The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
  • 10mM dNTPs
  • Forward primer (10μM)
  • Reverse primer (10μM)
  • Sterile H2O

Protocol

Make up the PCR mix (see below) and add 49μL to each well of a 96-well PCR plate or to each PCR tube and keep on ice. Pick a tiny amount (~1μL) for bacterial cells off of the plate with a sterile toothpick or similar. Mix thoroughly with the toothpick or up-and-down by pipette. It is very important that the cells are suspended in the PCR mix or it won't work.

  • If you want to freeze down or mini-prep the positive bacterial colonies the next day, after you dip your toothpick in the PCR mix you can then dip it into LB media (+ appropriate antibiotics) in a test tube and put that at 37°C on the roller drum. The next day you will have an overnight culture of bacteria appropriate for freezing down or mini-prepping.

Put the plate/tubes in the thermocycle and run the PCR program below. Once the thermocycler is finished, run out your products on a agarose gel with an appropriate DNA ladder.


PCR Reaction Mix

Reagent Volume
10x HotMaster Taq Buffer with Mg2+ 5μL
10mM dNTP mix 1μL
10μM Primer 1 1μL
10μM Primer 2 1μL
HotMaster Taq DNA polymerase 0.25μL
Sterile Water 41.75μL
Cells ~1μL
Total Volume 50μL

PCR Program

Run PCR on Colony PCR program :

  • 95°C 5min
  • Repeat the following three steps 30 times:
    • 94°C 45s
    • 50°C 1min
    • 68°C 1min 30s
  • 72°C 10min
  • 4°C Forever
    • Please don't leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Megan N McClean 15:07, 29 April 2012 (EDT): Adjust the parameters of the PCR program to be appropriate for your primers and desired product size (ie, shorter or lengthen the extension time, change the annealing temperature, etc).

*Megan N McClean 15:07, 29 April 2012 (EDT): We really don't need to be doing 50μL reactions to check colonies. If someone has the time, please try a smaller volume (20μL) of this protocol and see if it works and then update this protocol with a note.

  • Michael T. Patel 17:33, 26 April 2013 (EDT): Running reaction volumes of 25μL works fine, though you should use larger master mixtures and allotting at the end to avoid pipetting error with small volumes. Also you can probably use reaction volumes smaller than 25μL, though you may want to consider adding mineral oil to these.

References

Contact

Megan N McClean

or instead, discuss this protocol.

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