130
edits
Knight:Colony PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
m
→Notes
(→Notes: Added short note on minor protocol difference) |
m (→Notes) |
||
| Line 38: | Line 38: | ||
#Lately, I've been carrying out this procedure using a multichannel pipettor to speed steps up. I use a P50 multichannel pipettor to add 20μL water to the appropriate number of wells in a 96 well plate/ Picking colonies is essentially the same except that I inoculate the colony into a 96 well plate rather than a tube. The PCR is carried out in 8 tube strips rather than individual tubes. After adding 9.5μL of the primer + PCR supermix master mix to each PCR tube, I transfer 1μL of colony-water mixture to the reaction tube using a P10 multichannel pipettor. (I transfer 1 μL rather than 0.5μL because it is easier to pipette.) I then touch spin the PCR tube strips in a rack using a swinging bucket rotor in order to collect the contents of the tube to the bottom. The PCR conditions are the same. --[[Reshma Shetty | RS]] | #Lately, I've been carrying out this procedure using a multichannel pipettor to speed steps up. I use a P50 multichannel pipettor to add 20μL water to the appropriate number of wells in a 96 well plate/ Picking colonies is essentially the same except that I inoculate the colony into a 96 well plate rather than a tube. The PCR is carried out in 8 tube strips rather than individual tubes. After adding 9.5μL of the primer + PCR supermix master mix to each PCR tube, I transfer 1μL of colony-water mixture to the reaction tube using a P10 multichannel pipettor. (I transfer 1 μL rather than 0.5μL because it is easier to pipette.) I then touch spin the PCR tube strips in a rack using a swinging bucket rotor in order to collect the contents of the tube to the bottom. The PCR conditions are the same. --[[Reshma Shetty | RS]] | ||
# We use a [[Seth:Quick_Transformant_Screen|very similar protocol]] with different PCR conditions. Also we use half the DNA for our PCRs and add media to the remainder. Enough cells survive to innoculate overnight cultures for minipreps of which colonies you decide are needed. [[User:Seth|SK]] <small>''January 29th, 2007''</small> | # We use a [[Seth:Quick_Transformant_Screen|very similar protocol]] with different PCR conditions. Also we use half the DNA for our PCRs and add media to the remainder. Enough cells survive to innoculate overnight cultures for minipreps of which colonies you decide are needed. [[User:Seth|SK]] <small>''January 29th, 2007''</small> | ||
#In a normal PCR reaction setting, a template of 2μL from a 20μL + one colony mix (nicely vortexed) works great for ''S.aureus'', provided we load at least 10μL of the PCR reaction mix in the gel. [[User:Vijay|NV]] | |||
