Team:Michigan/Pili Expression
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==8/14/2010== | ==8/14/2010== | ||
''Kevin, Marc, Alena'' | ''Kevin, Marc, Alena'' | ||
- | + | '''Miniprep pBAD plasmid''' | |
- | + | #inoculate 5.0mL LB in 50mL conical tubes w/ 100ug/mL of ampilicin | |
+ | ##the cultures (2 of them) grew for ~12hrs (8am to 8pm) | ||
+ | #centrifuge (using the 50mL conical tubes) for 5000rpm for 10min at 4C | ||
+ | #carefully discard the supernatant and resuspend the pellet with 250uL of P1 buffer (kept in the 4C fridge) | ||
+ | #transfer into a labelled 1.5mL eppendorf tube (set pipetman to 500uL just in case) | ||
+ | #add 250uL P2 Buffer--> invert 4-6times (should turn blue)--> add 350uL N3 buffer--> invert 4-6times (should become vicious or clumpy) | ||
+ | ##centrifuge for 13,000rpm for 10mins | ||
+ | #pipet out supernatant into a QIA spin column--> centrifuge for 60s--> discard flow through | ||
+ | ##did not pipet out all of the supernatant | ||
+ | #add 500uL PB buffer --> centrifuge 60s--> discard flow through | ||
+ | #add 750uL PE buffer--> centrifuge 60s--> discard--> centrifuge 60s again | ||
+ | #transfer into a labeled 1.5mL eppendorf tube--> add 50uL EB (elution buffer) | ||
+ | ##allow it to sit for 1 min (it helps to release the DNA from the column) | ||
+ | |||
+ | '''fimB PCR product Purification''' | ||
#Used sample A and B of PCR product (save C and D for later) | #Used sample A and B of PCR product (save C and D for later) | ||
- | ##total volume | + | ##total volume of PCR product = 81 uL (40.5uL separately) |
- | ( | + | #add 5 volumes of PB buffer (202.5uL) to 1 volume of PCR product (40.5uL); invert |
+ | #transfer into a QIAquick spin column (provided in the Qiagen kit) | ||
+ | ##set pipetman to 260uL to be sure to get all of the mixture | ||
+ | #centrifuge spin column at 13,000rpm for 60s | ||
+ | #discard the flow through (in the collection tube) and add 750uL PE buffer (to wash the DNA) | ||
+ | repeat step 4 again | ||
+ | #discard flow through and centrifuge again to get the remain buffers out | ||
+ | #place the column into a labeled 1.5mL eppendorf tube | ||
+ | #add 50uL EB buffer (to elute out the DNA) directly to the white inner circle of the column (avoid touching the pipet tip to the column) | ||
+ | ##allow the mix to sit in the column for 1 min, then centrifuge for 1 min (13,000rpm) | ||
+ | ##remember to point the cap of the eppendorf tube in the opposite direction of the centrifuge machine | ||
+ | '''fimB Digest''' | ||
+ | #pre-programmed PCR machine to Digest (DIG1) | ||
+ | #use 5 PCR reaction tubes for each; 5 for fimB and 5 for pBAD | ||
+ | #add the following amounts in that order (total volume of 20uL) | ||
+ | **16 uL of the DNA (fimB and pBAD to their respective tubes) | ||
+ | **2 uL of NEB 2 buffer | ||
+ | **1 uL NcoI | ||
+ | #incubate for 37C overnight (12hrs) | ||
+ | ##place into 4C fridge the next day | ||
+ | **1uL HindIII | ||
| | | | ||
+ | |||
=='''In the Lab'''== | =='''In the Lab'''== | ||
[[Image:pili01.png|middle|250px]] | [[Image:pili01.png|middle|250px]] |
Revision as of 08:14, 16 August 2010