Team:Michigan/Pili August September
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PCR #1 | PCR #1 | ||
- | Used a gradient from | + | Used a gradient from 40ºC to 60ºC for the first 3 cycles to find the optimum annealing temperature. |
All of the annealing temperatures gave a good result according to the gel. | All of the annealing temperatures gave a good result according to the gel. | ||
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''Kevin, Marc, Alena'' | ''Kevin, Marc, Alena'' | ||
- | Used a | + | Used a 57ºC degree annealing temperature to get enough DNA for the digest and ligation. |
4 out of the 5 PCR reactions worked well according to the gel. | 4 out of the 5 PCR reactions worked well according to the gel. | ||
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4. Stabbed cryostock using a sterile 200 microliter pipette tip and pipetted into media from step 2. | 4. Stabbed cryostock using a sterile 200 microliter pipette tip and pipetted into media from step 2. | ||
- | 5. At 8:05PM placed the two falcon tubes from step 4 into the incubator/shaker at | + | 5. At 8:05PM placed the two falcon tubes from step 4 into the incubator/shaker at 30ºC. |
==8/14/2010== | ==8/14/2010== | ||
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'''Miniprep pBAD plasmid''' | '''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 4ºC |
- | # | + | #Carefully discard the supernatant and resuspend the pellet with 250uL of P1 buffer (kept in the 4ºC 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 at 13,000 rpm for 10 min |
- | # | + | #Pipette out supernatant into a QIA spin column--> centrifuge for 60s--> discard flow through |
##did not pipet out all of the supernatant | ##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''' | '''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 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''' | '''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) | *16 uL of the DNA (fimB and pBAD to their respective tubes) | ||
*2 uL of NEB 2 buffer | *2 uL of NEB 2 buffer | ||
*1 uL NcoI | *1 uL NcoI | ||
*1uL HindIII | *1uL HindIII | ||
- | # | + | #Incubate for 37ºC overnight (12hrs) |
- | ## | + | ##Place into 4ºC fridge the next day |
==8/16/2010== | ==8/16/2010== | ||
''Kevin, Marc, Alena'' | ''Kevin, Marc, Alena'' | ||
- | Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store | + | Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store. |
Attempted experiment: | Attempted experiment: | ||
*Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD) | *Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD) | ||
**CIP will cut off the 3' phosphate to prevent the plasmid from folding back on itself | **CIP will cut off the 3' phosphate to prevent the plasmid from folding back on itself | ||
- | * | + | *Incubate CIP-pBAD at 37C for 1 hr and then heat-shock (65ºC for 15 min) |
'''We made a huge human error when setting up our thermal cycle program on the PCR machine. We did not realize we only set the thermal cycle for our digest on 8/14/2010>> [https://2010.igem.org/Team:Michigan/Pili_Expression#8.2F14.2F2010] for 12 minutes (12:00) rather than the intended time period of 12 hours (12:00:00). This explains the condensation present in the PCR machine (it stayed at 4C for too long). We discovered our mistake after running the incubation of CIP+pBAD for 37C for (1:00) 1 minute then heat-shock at 65C for 15 minute. It is VERY IMPORTANT to triple check one another when entering in any program.''' | '''We made a huge human error when setting up our thermal cycle program on the PCR machine. We did not realize we only set the thermal cycle for our digest on 8/14/2010>> [https://2010.igem.org/Team:Michigan/Pili_Expression#8.2F14.2F2010] for 12 minutes (12:00) rather than the intended time period of 12 hours (12:00:00). This explains the condensation present in the PCR machine (it stayed at 4C for too long). We discovered our mistake after running the incubation of CIP+pBAD for 37C for (1:00) 1 minute then heat-shock at 65C for 15 minute. It is VERY IMPORTANT to triple check one another when entering in any program.''' | ||
Rest of the day: | Rest of the day: | ||
- | * | + | *Headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4ºC fridge |
==8/17/2010== | ==8/17/2010== | ||
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Met with Chris to discuss about yeast agglutination | Met with Chris to discuss about yeast agglutination | ||
- | *Chris sent us a possible paper with a decent protocol to look at for the assay [[Media:YeastAgglutinationPaper.pdf]] | + | *Chris sent us a possible paper with a decent protocol to look at for the assay [[Media:YeastAgglutinationPaper.pdf]]. |
'''NOTE ABOUT K.O. STRAINS''' | '''NOTE ABOUT K.O. STRAINS''' | ||
*fimE K.O. did not grow out (which contradicts the prediction that fimE K.O grows faster than fimB K.O.) | *fimE K.O. did not grow out (which contradicts the prediction that fimE K.O grows faster than fimB K.O.) | ||
- | *try letting the plate grow at | + | *try letting the plate grow at 37ºC for a longer period of time (one colony observed) |
*remove the filter circle paper and put onto a new LB-agar plate | *remove the filter circle paper and put onto a new LB-agar plate | ||
*suspect too much kanamycin on the plate | *suspect too much kanamycin on the plate | ||
Lab work: | Lab work: | ||
- | #add CIP (calf intestine phosphatase= cuts off the 3' phosphate to prevent plasmid from closing back on itself) to plasmid (pBAD) reaction tubes only--> incubate at | + | #add CIP (calf intestine phosphatase= cuts off the 3' phosphate to prevent plasmid from closing back on itself) to plasmid (pBAD) reaction tubes only--> incubate at 37ºC for 1 hour (using PCR machine) |
#heat/inactivate pBAD and fimB reaction tubes for 15 min at 65C (again using PCR machine) | #heat/inactivate pBAD and fimB reaction tubes for 15 min at 65C (again using PCR machine) | ||
#perform DNA purification on the digests (follow 8/16/2010 procedure) | #perform DNA purification on the digests (follow 8/16/2010 procedure) | ||
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Ran Ligation of FimB and pBAD for 16 hrs O/N | Ran Ligation of FimB and pBAD for 16 hrs O/N | ||
- | Prepared culture of | + | Prepared culture of DH5α for electroporation tomorrow. |
==8/23/2010== | ==8/23/2010== | ||
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#Ligation product | #Ligation product | ||
#500 ul butanol | #500 ul butanol | ||
- | Spin at | + | Spin at 4ºC, 13000 rpm for 20 min |
Made ampicillin plates | Made ampicillin plates | ||
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''Kevin'' | ''Kevin'' | ||
- | Created frozen stocks of K12 and ΔFim::kan w/the plasmid from overnight cultures. Stored them in box 1 in the ERB - | + | Created frozen stocks of K12 and ΔFim::kan w/the plasmid from overnight cultures. Stored them in box 1 in the ERB -20ºC freezer. |
Ran miniprep of K12 and ΔFim::kan, using 5 ml of each culture. | Ran miniprep of K12 and ΔFim::kan, using 5 ml of each culture. | ||
Note: Centrifuge in ERB will only go up to 5000 rpm w/50 ml tubes, therefore we transferred the culture to several eppendorf tubes. This was probably not a good idea, because we were left with a lot of leftover supernatant and that could have diluted the buffers. In the future, we should centrifuge the culture 1 ml at a time, and add 1 ml after each cycle. | Note: Centrifuge in ERB will only go up to 5000 rpm w/50 ml tubes, therefore we transferred the culture to several eppendorf tubes. This was probably not a good idea, because we were left with a lot of leftover supernatant and that could have diluted the buffers. In the future, we should centrifuge the culture 1 ml at a time, and add 1 ml after each cycle. | ||
- | Stored the product from miniprep in box 2 in the ERB - | + | Stored the product from miniprep in box 2 in the ERB -20ºC freezer. |
==9/9/2010== | ==9/9/2010== |
Latest revision as of 03:05, 27 October 2010