PrbbBB:colony pcr v1: Difference between revisions
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==Overview== | ==Overview== | ||
A colony PCR protocol for screening up to 8 clones each for up to 12 different assembly reactions in parallel. Colonies are picked into a 96-well plate with water. The PCR reactions are set up in another 96 well plate and the template DNA is copied over with a multi-channel pipette. During the colony picking, a copy of each clone is put into a deepwell plate with liquid medium which is later used to inoculate over-night cultures for miniprep and cell stock. | |||
==Materials== | ==Materials== | ||
*96-well PCR plate | * two sterile 96-well PCR plates | ||
*AmpliTaq DNA Polymerase 5 U/µl (Roche) | * two sterile 96-deepwell plates (preferably 2 ml volume/square well) | ||
*AmpliTaq Buffer 10 x | * adhesive tape for plate sealing | ||
*dNTP mix 10mM each nucleotide | * gas-transmissible adhesive tape for deepwell plate sealing | ||
*ddH2O | * 12-channel 1-10µl pipette | ||
*primers: | * multi-dispensing pipette (2 µl minimum) + sterile tips | ||
**[http://partsregistry.org/wiki/index.php/Part:BBa_G00100 BBa_G00100 (VF2)] [http://brickit.crg.es/registry/biobrick/5/ CRG-internal registry] | * AmpliTaq DNA Polymerase 5 U/µl (Roche) | ||
**[http://partsregistry.org/wiki/index.php/Part:BBa_G00101 BBa_G00101 (VR)] [http://brickit.crg.es/registry/biobrick/6/ CRG-internal registry] | * AmpliTaq Buffer 10 x | ||
* dNTP mix 10mM each nucleotide | |||
* sterile ddH2O | |||
* primers: | |||
** [http://partsregistry.org/wiki/index.php/Part:BBa_G00100 BBa_G00100 (VF2)] [http://brickit.crg.es/registry/biobrick/5/ CRG-internal registry] | |||
** [http://partsregistry.org/wiki/index.php/Part:BBa_G00101 BBa_G00101 (VR)] [http://brickit.crg.es/registry/biobrick/6/ CRG-internal registry] | |||
* liquid LB or 2xTY medium with appropriate antibiotic (Chloramphenicol, Kanamycin, or Tetracycline depending on target plasmid) | |||
==Procedure== | ==Procedure== | ||
''' | '''I PCR reaction''' | ||
<table> | <table> | ||
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<table frame=box> | <table frame=box> | ||
<tr align=right> | <tr align=right> | ||
<td></td> <th width=100> | <td></td> <th width=100>11µl single reaction</th> <th width=100>60xMaster</th> <th width=100>115xMaster</th></tr> | ||
<tr align=right> <td>H2O</td> | <tr align=right> <td>H2O</td> <td>7.5µl</td> <td>450</td> <td>862</td> </tr> | ||
<tr align=right> <td> | <tr align=right> <td>10x Amplitaq buffer</td> <td>1.1µl</td> <td>66</td> <td>126.5</td> </tr> | ||
<tr align=right> <td>10mM dNTP</td> | <tr align=right> <td>10mM dNTP</td> <td>0.22µl</td> <td>13.2</td> <td>25.3</td> </tr> | ||
<tr><td></td></tr> | <tr><td></td></tr> | ||
<tr align=right> <td> | <tr align=right> <td>VF2 primer 100 µM</td> <td>0.55µl</td> <td>33</td> <td>63.3</td> </tr> | ||
<tr align=right> <td> | <tr align=right> <td>VR primer 100 µM</td> <td>0.55µl</td> <td>33</td> <td>63/3</td> </tr> | ||
<tr align=right> <td> | <tr align=right> <td>AmpliTaq 5U/µl</td> <td>0.1µl</td> <td>6</td> <td>11.5</td> </tr> | ||
<tr><td></td></tr> | <tr><td></td></tr> | ||
<tr align=right> <td>template DNA</td> | <tr align=right> <td>template DNA</td> <td>1µl</td> <td>--</td> <td>--</td> </tr> | ||
</table> | </table> | ||
</td> | </td> | ||
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<table frame=box> | <table frame=box> | ||
<tr><th></th> <th>PCR Program</th> | <tr><th></th> <th>PCR Program</th> | ||
<tr><td></td> <td> 10' @95°C </td></tr> | |||
<tr> | <tr><td>30 × </td> <td> (30" @95°C; 15" @64; ''t<sub>ext</sub>''@72°C); </td></tr> | ||
<tr><td></td> <td> optional: 10' @72°C </td></tr> | |||
<tr><td> | <tr><td></td> <td> ∞ 4°C </td></tr> | ||
<tr><td></td> | |||
</table> | </table> | ||
* extension time '''t<sub>ext</sub>''' = (kb insert length) × | * extension time '''t<sub>ext</sub>''' = (kb insert length) × 1' (1 min) | ||
</td><tr> | </td><tr> | ||
</table> | </table> | ||
# prepare 96-well "Lysis" (PCR) plate: | |||
## sterilize plate and pipette tips | |||
## multi-dispense 50 µl H2O into each well | |||
# prepare 96-deepwell "Copy" plate: | |||
## sterilize plate and pipette tips | |||
## multi-dispense 100-200 µl sterile LB + appropriate antibiotic into each well | |||
# colony picking: | |||
## pick colonies of one assembly with sterile 10 µl tips into... | |||
## ... the wells of one column (like A1-H1 or A2-H2) on the Lysis plate | |||
## pipette up and down | |||
## eject each tip into the same position on the "Copy" deepwell plate | |||
## proceed to next column for next assembly | |||
# seal the "Copy" deepwell plate, shake vigorously for 1 min and let it stand at room temperature | |||
# prepare PCR plate: | |||
## multi-dispense 10 µl PCR mastermix into each well | |||
## copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette) | |||
## seal PCR plate with adhesive tape | |||
# run PCR program | |||
'''II Agarose Gel''' | '''II Agarose Gel''' | ||
# | # prepare a large 0.7 - 1.5 % Agarose gel (depending on fragment size) | ||
# | # multi-dispense 2 µl loading buffer into each well of the PCR plate | ||
# load gel with multi-channel pipette | |||
# run, analyze, enjoy! | |||
'''III Rescue positive clones''' | |||
# inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB) | |||
# seal plate with gas-transmissible adhesive tape | |||
# grow over night with vigorous shaking (700 r.p.m.) at 37°C | |||
==Notes== | ==Notes== |
Latest revision as of 10:10, 11 February 2010
Overview
A colony PCR protocol for screening up to 8 clones each for up to 12 different assembly reactions in parallel. Colonies are picked into a 96-well plate with water. The PCR reactions are set up in another 96 well plate and the template DNA is copied over with a multi-channel pipette. During the colony picking, a copy of each clone is put into a deepwell plate with liquid medium which is later used to inoculate over-night cultures for miniprep and cell stock.
Materials
- two sterile 96-well PCR plates
- two sterile 96-deepwell plates (preferably 2 ml volume/square well)
- adhesive tape for plate sealing
- gas-transmissible adhesive tape for deepwell plate sealing
- 12-channel 1-10µl pipette
- multi-dispensing pipette (2 µl minimum) + sterile tips
- AmpliTaq DNA Polymerase 5 U/µl (Roche)
- AmpliTaq Buffer 10 x
- dNTP mix 10mM each nucleotide
- sterile ddH2O
- primers:
- liquid LB or 2xTY medium with appropriate antibiotic (Chloramphenicol, Kanamycin, or Tetracycline depending on target plasmid)
Procedure
I PCR reaction
|
| ||||||||||||||||||||||||||||||||||||||||||||
- prepare 96-well "Lysis" (PCR) plate:
- sterilize plate and pipette tips
- multi-dispense 50 µl H2O into each well
- prepare 96-deepwell "Copy" plate:
- sterilize plate and pipette tips
- multi-dispense 100-200 µl sterile LB + appropriate antibiotic into each well
- colony picking:
- pick colonies of one assembly with sterile 10 µl tips into...
- ... the wells of one column (like A1-H1 or A2-H2) on the Lysis plate
- pipette up and down
- eject each tip into the same position on the "Copy" deepwell plate
- proceed to next column for next assembly
- seal the "Copy" deepwell plate, shake vigorously for 1 min and let it stand at room temperature
- prepare PCR plate:
- multi-dispense 10 µl PCR mastermix into each well
- copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette)
- seal PCR plate with adhesive tape
- run PCR program
II Agarose Gel
- prepare a large 0.7 - 1.5 % Agarose gel (depending on fragment size)
- multi-dispense 2 µl loading buffer into each well of the PCR plate
- load gel with multi-channel pipette
- run, analyze, enjoy!
III Rescue positive clones
- inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB)
- seal plate with gas-transmissible adhesive tape
- grow over night with vigorous shaking (700 r.p.m.) at 37°C
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: no comment
References
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or instead, discuss this protocol.