PrbbBB:colony pcr v1: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 43: | Line 43: | ||
<tr><td></td> <td> 10' @95°C </td></tr> | <tr><td></td> <td> 10' @95°C </td></tr> | ||
<tr><td> | <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></td> <td> optional: 10' @72°C </td></tr> | ||
<tr><td></td> <td> ∞ 4°C </td></tr> | <tr><td></td> <td> ∞ 4°C </td></tr> | ||
Line 53: | Line 53: | ||
</table> | </table> | ||
# prepare 96-well "Lysis" plate: | |||
## multi-dispense 50 µl H2O into each well | |||
## pick 8 colonies of one assembly into one column (like A1-H1 or A2-H2) | |||
## copy 4 µl per well from this row onto a backup LB plate with Ampicilin (using a 8-channel pipette) | |||
## proceed to next row for next assembly | |||
# prepare PCR plate: | |||
## multidispense 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 | |||
# put backup plates @ 37 °C | |||
'''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! | |||
==Notes== | ==Notes== |
Revision as of 05:18, 26 February 2009
Overview
Materials
- 96-well PCR plate
- AmpliTaq DNA Polymerase 5 U/µl (Roche)
- AmpliTaq Buffer 10 x
- dNTP mix 10mM each nucleotide
- ddH2O
- primers:
Procedure
I PCR reaction
|
| ||||||||||||||||||||||||||||||||||||||||||||
- prepare 96-well "Lysis" plate:
- multi-dispense 50 µl H2O into each well
- pick 8 colonies of one assembly into one column (like A1-H1 or A2-H2)
- copy 4 µl per well from this row onto a backup LB plate with Ampicilin (using a 8-channel pipette)
- proceed to next row for next assembly
- prepare PCR plate:
- multidispense 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
- put backup plates @ 37 °C
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!
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
raik: no comment
References
Contact
or instead, discuss this protocol.