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- | {|style="color:#1c2bf2;background-color:#fafa19;font-size:9pt;text-align:center" cellpadding="5" cellspacing="0" border="1" bordercolor="#fff" width="62%"
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- | !
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- | |Sunday
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- | |Monday
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- | |Tuesday
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- | |Wednesday
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- | |Thursday
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- | |Friday
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- | |Saturday
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- | !Week 1
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- | | -
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- | |[[Team:Michigan/Oil_Sands#6/28/2010|6/28/2010]]
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- | |[[Team:Michigan/Oil_Sands#6/29/2010|6/29/2010]]
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- | |[[Team:Michigan/Oil_Sands#6/29/2010 and 6/30/2010|6/30/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/1/2010|7/1/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/2/2010|7/2/2010]]
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- | |-
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- | !Week 2
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- | |[[Team:Michigan/Oil_Sands#7/7/2010|7/7/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/8/2010|7/8/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/9/2010|7/9/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/10/2010|7/10/2010]]
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- | |-
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- | !Week 3
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- | |[[Team:Michigan/Oil_Sands#7/12/2010|7/12/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/13/2010|7/13/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/14/2010|7/14/2010]]
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- | | -
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- | |-
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- | !Week 4
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- | | -
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- | !Week 5
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- | |[[Team:Michigan/Oil_Sands#7/25/2010|7/25/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/26/2010|7/26/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/27/2010|7/27/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/28/2010|7/28/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/29/2010|7/29/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/30/2010|7/30/2010]]
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- | |[[Team:Michigan/Oil_Sands#7/31/2010|7/31/2010]]
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- | |-
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- | !Week 6
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- | |[[Team:Michigan/Oil_Sands#8/1/2010|8/1/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/2/2010|8/2/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/3/2010|8/3/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/4/2010|8/4/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/5/2010|8/5/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/6/2010|8/6/2010]]
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- | |[[Team:Michigan/Oil_Sands#8/7/2010|8/7/2010]]
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- | |-
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- | |}
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- | ==Oil Sands Lab Notebook==
| |
| | | |
- | This team includes [[User:annlesn|Ann Lesnefsky]], [[User:rajabiab|Bryce Rajabian]], and [[User:kilholee|Kilho Lee]].
| |
| | | |
- | ==6/28/2010== | + | ==Oil Sands Lab Notebook== |
- | ''' ''Pseudomonas putida'' KT2440 Antibiotic Resistance'''
| + | |
- | *[[Media:6-28-2010_Protocol_for_making_cultures_from_a_-80C_freezer_stock.pdf|Protocol for starting overnight culture from -80°C freezer]]
| + | |
- | *All protocols can also be found in the ''Protocols'' section of the Notebook
| + | |
- | | + | |
- | ==6/29/2010==
| + | |
- | ''' ''Pseudomonas putida'' KT2440 Antibiotic Resistance'''
| + | |
- | *[[Media:6-29-2010_Making_Frozen_Stoks.pdf|Protocol for making frozen stocks]]
| + | |
- | | + | |
- | ==6/29/2010 and 6/30/2010==
| + | |
- | ''' ''Pseudomonas putida'' KT2440 Antibiotic Resistance'''
| + | |
- | *[[Media:6-27-2010_P._putida_antibiotic_resistance_protocol.pdf|Protocol for ''P. putida'' KT2440 antibotic resistance]]
| + | |
- | | + | |
- | ==7/1/2010==
| + | |
- | ''' ''Literature Review'' Napthenic Acids Composition & HPLC Analysis'''
| + | |
- | *[[Media:6-29-2010_Naphthenic_acids_Composition.pdf|Literature Review of the composition of napthenic acids in oil sands]]
| + | |
- | | + | |
- | ==7/2/2010==
| + | |
- | *[[Media:6-30-10_HPLC_Column_Comparison_Sheet1.pdf|Literature Review of HPLC columns for analysis of napthenic acids]]
| + | |
- | ''' ''Pseudomonas putida'' KT2440 Antibiotic Resistance'''
| + | |
- | *[[Media:7-2-2010_Results_P._putida_antibiotic_resistance.pdf|Results]]
| + | |
- | ==7/7/2010==
| + | |
- | ''Ann''
| + | |
- | | + | |
- | '''Biobrick Transformation''' with Alex and Jennifer
| + | |
- | *Made CaCl2 solution, and 100 mg/mL amphicilin stock solution according to the media section of the wiki
| + | |
- | *Started an overnight culture of DH5alpha according to the heat shock transformation protocol
| + | |
- | **A 12 mL culture was started because we were multiplying the protocol by 4
| + | |
- | | + | |
- | ==7/8/2010==
| + | |
- | ''Ann''
| + | |
- | | + | |
- | '''Biobrick Transformation''' with Marc, Katie and Audra according to the heat shock transformation protocol
| + | |
- | *Started culture for biobrick transformation from over night at 2:30
| + | |
- | *At 5:00pm the cultures had overgrown to an OD600 of 1.2
| + | |
- | **A a 1:3 dilution of cells was performed and the cells were allowed to go through another doubling period of 20 minutes
| + | |
- | *The OD600 was measured again and found to be around 0.500
| + | |
- | *the cultures were placed on ice at 5:30 and allowed to chill for 20 minutes
| + | |
- | *The washings were performed and the comp cells were resuspended in the residual CaCl2 only
| + | |
- | *The OD600 of the comp cells without the glycerol was above 0.3 therefore the comp cells were concentrated enough
| + | |
- | *The heat shock was performed for the following biobricks
| + | |
- | **BBa_179015
| + | |
- | **BBa_179005
| + | |
- | **BBa_145001
| + | |
- | **BBa_117008
| + | |
- | **BBa_117002
| + | |
- | **BBa_103006
| + | |
- | *The cultures were placed in the 30C shaker to grow up for an hour at 30C at 9:00pm
| + | |
- | *The cultures were plated at 10:00pm
| + | |
- | ==7/9/2010==
| + | |
- | ''Ann''
| + | |
- | | + | |
- | '''Biobrick Transformation''' with Josh, Prae, Charlie according to the miniprep protocol
| + | |
- | *After 9 hours of growing at 37C, the plates did not have any colonies on them
| + | |
- | *After 22 hours of growth plates that had grown had clear colonies
| + | |
- | **The positive and negative controls grew out accordingly
| + | |
- | **Only BBa_179015 and BBa_179005 had colonies (both from plate 1 and none from plate 2...)
| + | |
- | ***BBa_179015 had over 100 colonies
| + | |
- | ***BBa_179005 had 2 colonies
| + | |
- | *5 mL overnight cultures were started with 100 mg/mL amp at 8pm from a single colony on each plate
| + | |
- | | + | |
- | ==7/10/2010==
| + | |
- | ''Ann''
| + | |
- | | + | |
- | '''Biobrick Transformation''' with Marcus, Bryce, Kilho, Josh, Charlie, Jeremy
| + | |
- | | + | |
- | Miniprep
| + | |
- | *Made frozen stocks from overnight culture of miniprep
| + | |
- | *Performed miniprep on BBa_179015 and BBa_179005
| + | |
- | *Measured DNA concentration with the Nanodrop
| + | |
- | **BBa_179015-11 ng/mL
| + | |
- | **BBa_179005-40 ng/mL
| + | |
- | | + | |
- | Digest
| + | |
- | *Ran according to the digest protocol in the protocol section
| + | |
- | **Cut parts with EcoRI and PstI
| + | |
- | *Digested at 37C for 30 minutes
| + | |
- | | + | |
- | Gel
| + | |
- | *Ran according to the protocol in the protocol section
| + | |
- | *Used 8 well comb instead of 13 well comb so the samples were too dilute too see
| + | |
- | | + | |
- | ==7/12/2010==
| + | |
- | | + | |
- | ''Ann''
| + | |
- | | + | |
- | '''Biobrick Transformation''' with Jeremy
| + | |
- | | + | |
- | Gel
| + | |
- | | + | |
- | [[Image:7-12-2010_T7_promoter_and_T7_gfp_biobricks.JPG|300px]]
| + | |
- | *Reran gel from 7/10/2010
| + | |
- | **Lane 1-Invitrogen 1 kb plus ladder
| + | |
- | **Lane 2-Digested Bba_179005
| + | |
- | ***Ran out of undigested Bba_179005
| + | |
- | **Lane 3-Digested Bba_179015
| + | |
- | **Lane 4-Uncut miniprep plasmid Bba_179015
| + | |
- | | + | |
- | We found that we successfully extracted BBa_179015, T7-GFP, because there were expected bands at 906 and 2079 as faintly seen in the gel picture above. No appeared for biobrick Bba_179005, T7 promoter, so this biobrick will be transformed again.
| + | |
- | | + | |
- | '''Biobrick Transformation take 2''' with Jeremy
| + | |
- | | + | |
- | Autoclaved DI water for transformation and autoclaved sterile containers
| + | |
- | *to autoclave sterile containers fill with DI water and decant the water before you start the culture in the container
| + | |
- | | + | |
- | ==7/13/2010==
| + | |
- | | + | |
- | '''Biobrick Transformation take 2''' with Jeremy, Marcus, Josh, Kevin, Audra, Katie
| + | |
- | | + | |
- | ''Ann''
| + | |
- | | + | |
- | Performed according to the electroporation transformation
| + | |
- | *Started the culture at 9:05am
| + | |
- | *Removed the culture at 12:05pm with an OD600 of 0.809
| + | |
- | *The OD600 of the comp cells were 1.2 after washing
| + | |
- | *The time constant for all electroporation were between 5.6 and 5.8 for the following biobricks
| + | |
- | **Bba_K117008
| + | |
- | **Bba_K117008 #2 (from resuspending remaining part left in registry in 15 uL of ultra pure water)
| + | |
- | **Bba_K117002
| + | |
- | **Bba_K145001
| + | |
- | **Bba_K103006
| + | |
- | **Bba_I719005
| + | |
- | *The cultures were allowed to grow in the incubator from 2:15-4:00pm
| + | |
- | **The cultures started to clump after growing this time
| + | |
- | *100 uL of cells were plated on 100 mg/mL AMP plates
| + | |
- | | + | |
- | ==7/14/2010==
| + | |
- | | + | |
- | '''Biobrick Transformation take 2'''
| + | |
- | | + | |
- | ''Ann''
| + | |
- | | + | |
- | Electroporation Transformation
| + | |
- | | + | |
- | All of the transformation plates grew out! (minus the negative control of course)
| + | |
- | | + | |
- | This means we should do all transformations by electroporation from now on. Just check with the Lin Lab to make sure we can use the electroporation machine for a few hours before starting
| + | |
- | | + | |
- | Miniprep
| + | |
- | *Started overnight cultures in 5 mL of LB plus 100 mg/mL AMP
| + | |
- | **The following biobricks were started from the transformation plate
| + | |
- | ***Bba_K117008
| + | |
- | ***Bba_K117002
| + | |
- | ***Bba_K145001
| + | |
- | ***Bba_K103006
| + | |
- | ***Bba_I719005
| + | |
- | **The following biobricks were started from the frozen stock in the -80C freezer
| + | |
- | ***BBa_K719015
| + | |
- | | + | |
- | '''INPNC Biobrick part'''
| + | |
- | *We received the INPNC biobrick (Bba_K265008) from UC Davis 2009 team. THANK YOU!!!
| + | |
- | *It was shipped on LB plates in a pMA-SK plasmid from Mr. Gene in ''E. coli'' DH5alpha
| + | |
- | *Since this plasmid has AMP antibiotic resistance I am pouring plates today with Marc and Kevin with 100 mg/mL AMP resistance to streak out the culture tomorrow with Josh
| + | |
- | | + | |
- | ==7/25/2010==
| + | |
- | | + | |
- | '''Biobrick Transformation of suface display and pBAD'''
| + | |
- | | + | |
- | ''Ann''
| + | |
- | | + | |
- | Tonight I started an 8mL culture of E. coli DH5alpha in LB broth and place in the 30C shaker to grow out overnight
| + | |
- | | + | |
- | ==7/26/2010==
| + | |
- | | + | |
- | '''Biobrick Transformation of suface display and pBAD'''
| + | |
- | | + | |
- | ''Ann''
| + | |
- | | + | |
- | Electroporation transformation
| + | |
- | | + | |
- | Started larger culture for comp cell preparation in 40mL of LB in a 500 mL sterile container at 9:00am
| + | |
- | | + | |
- | At 11:30am the OD of the culture was 0.730 and the cultures were placed on ice
| + | |
- | | + | |
- | The transformation of the biobricks was performed for according to the electroporation transformation protocol listed under the protocol section
| + | |
- | | + | |
- | The biobricks removed from the registry are as follows
| + | |
- | *E0040 (GFP)
| + | |
- | *K157013 (linker)
| + | |
- | *I0500 (pBAD)
| + | |
- | | + | |
- | The miniprep of I79015 was used as a positive control and a negitive control was also run
| + | |
- | | + | |
- | The OD600 of the comp cells were not measured
| + | |
- | | + | |
- | All of the time constants for the transformation were above 5
| + | |
- | | + | |
- | the GFP, linker parts and the positive and negitive control were plated on 100 mg/mL AMP plates. The pBAD part and negitive control were plated on 50 mg/mL KAN
| + | |
- | | + | |
- | ==7/27/2010==
| + | |
- | | + | |
- | '''Biobrick Transformation of suface display and pBAD'''
| + | |
- | | + | |
- | Transformation Results
| + | |
- | | + | |
- | The GFP and linker transformation worked and many colonies grew out on the 100 mg/mL AMP plates
| + | |
- | | + | |
- | The pBAD part did not grow out on the KAN plates. Upon further inspection this plasmid needs to be grown with IPTG to switch the origin of replication to a high copy above 100 copies per cell. Otherwise, another orgin of replication dominates that is less than 10 copies per cell and the antibiotic concentration of 50 mg/mL maybe too high. This transformation will have to be tried again
| + | |
- | | + | |
- | '''Miniprep of surface display'''
| + | |
- | | + | |
- | At 9:00am a 5 mL culture of the newly transformed GFP part, the newly transformed linker part, the ompA part transformed previously and the INP from the UC davis team were started for a miniprep in LB with 100 mg/mL AMP.
| + | |
- | | + | |
- | The modified miniprep protocol was used listed on the protocol section of the wiki to get a higher DNA concentration
| + | |
- | | + | |
- | At 9:00pm the GFP, linker and INP parts were miniprepped. The ompA culture had not grown out (b/c not enough -80C freezer stock was added) and a new culture was restarted at 11:00pm in 5 mL of LB with 100 mg/mL AMP.
| + | |
- | | + | |
- | '''Pouring Plates'''
| + | |
- | | + | |
- | More LB with 100 mg/mL AMP plates and LB with 25 mg/mL KAN plates were poured
| + | |
- | | + | |
- | ==7/28/2010==
| + | |
- | | + | |
- | '''OmpA Miniprep'''
| + | |
- | | + | |
- | The OmpA culture grew out and was miniprepped according the the modified miniprep protocol at 11:30am
| + | |
- | | + | |
- | '''Nanodrop of surface display parts'''
| + | |
- | | + | |
- | The minipreped cultures from yesterday were nanodropped to determine the DNA concentration according to the protocol in the protocol section of the wiki
| + | |
- | *GFP: 47.8 ng/uL
| + | |
- | *linker: 33.2 ng/uL
| + | |
- | *INP: 41.8 ng/uL
| + | |
- | *OmpA: 49.3 ng/uL
| + | |
- | | + | |
- | '''Digest of surface display parts'''
| + | |
- | | + | |
- | The following biobricks were digested according to the protocol on the wiki with the enzymes listed below
| + | |
- | | + | |
- | *GFP 1: XbaI and PstI
| + | |
- | *GFP 2: EcoRI and XbaI
| + | |
- | *INP 1: EcoRI and SpeI
| + | |
- | *INP 2: SpeI and PstI
| + | |
- | *Linker 1: XbaI and PstI
| + | |
- | *Linker 2: SpeI and PstI
| + | |
- | *OmpA 1: EcoRI and SpeI
| + | |
- | *OmpA 2: SpeI and PstI
| + | |
- | | + | |
- | '''Gel of Digest for surface display parts'''
| + | |
- | | + | |
- | A gel was run of the above digest with uncut plasmid as a control
| + | |
- | | + | |
- | [[Image:7-28biobrickgel.jpg|300px]]
| + | |
- | | + | |
- | *Lane 1: invitrogen 1 kb plus ladder
| + | |
- | *Lane 2: GFP cut with XbaI and PstI (GFP: 720 bp Backbone: 2079 bp)
| + | |
- | *Lane 3: GFP cut with EcoRI and XbaI
| + | |
- | *Lane 4: uncut GFP plamid
| + | |
- | *Lane 5:INP cut with EcoRI and SpeI (INP: 924 bp backbone: 2550 bp)
| + | |
- | *Lane 6:INP cut with SpeI and PstI
| + | |
- | *Lane 7:uncut INP plasmid
| + | |
- | *Lane 8:Linker cut with XbaI and SpeI (Linker: 45 bp backbone: 2428 bp)
| + | |
- | *Lane 9:Linker cut with SpeI and PstI
| + | |
- | *Lane 10: uncut Linker plasmid
| + | |
- | *Lane 11: OmpA cut with EcoRI and SpeI (OmpA: 464 bp backbone: 2079 bp)
| + | |
- | *Lane 12: OmpA cut with SpeI and PstI
| + | |
- | *Lane 13:uncut OmpA plasmid
| + | |
- | | + | |
- | ==7/29/2010==
| + | |
- | | + | |
- | '''Biofilm Assay in LB media'''
| + | |
- | | + | |
- | Started cultures of E. coli K12, P. putida (oilsands) and P. fluorescens (oilsands) in 2 mL of LB and grew in the 30C shaker overnight
| + | |
- | | + | |
- | ==7/30/2010==
| + | |
- | | + | |
- | '''Biofilm Assay in LB media'''
| + | |
- | | + | |
- | Today we carried out the following [[Media:7-28-2010_Biofilm_Formation_Experiment.pdf|biofilm assay protocol]]
| + | |
- | | + | |
- | *@ 8:10am the cultures were started from the overnight
| + | |
- | *@ 10:00am the OD600 of each culture was measured to be
| + | |
- | **E. coli K12: 0.8
| + | |
- | **P. fluorescens: 1.2
| + | |
- | **P. putida: 1.0
| + | |
- | ***These cultures were overgrown but still used for the experiment
| + | |
- | | + | |
- | The results of this experiment are presented in the chart below
| + | |
- | | + | |
- | [[Image:7-30-2010_Biofilm_formation_in_LB_media_jpeg.jpg|500px]]
| + | |
- | | + | |
- | There is a large error in the P. putida strain because there was the largest variation in the OD 600 of the liquid culture which also resulted in a large variation in the CV OD 600 reading
| + | |
- | | + | |
- | We need to find a reference to compare our biofilms to. This could be either a strain that does not form biofilms or a strain that forms biofilms very well
| + | |
- | | + | |
- | '''Transformation of pBAD'''
| + | |
- | | + | |
- | Marcus and Alex tried the transformation of pBAD again according to the electroporation protocol with Ann's Notes
| + | |
- | | + | |
- | The recovery after electroporation was performed in 1 mL of LB with 1mM of IPTG added in order to have the pBAD part have a high copy number
| + | |
- | | + | |
- | 30 uL of 1M IPTG solution was added to a 25 mg/mL KAN plate and spread with sterile beads in order to keep the high copy plasmid in the pBAD part while plating
| + | |
- | | + | |
- | ==7/30/2010==
| + | |
- | | + | |
- | '''Transformation of pBAD'''
| + | |
- | | + | |
- | The transformation was successful and there were about 30 colonies on the plate. One colony was picked at 7:30 pm for a miniprep the next day and added to 5mL of LB + 25 mg/mL KAN + 1 mM IPTG and grown out in the 30C shaker
| + | |
- | | + | |
- | Another culture was started of GFP from the -80C freezer stock in 5 mL of LB + 100 mg/mL AMP for a miniprep. Both the ligations with GFP did not work, so as long as we are miniprepping and digesting we want to re-verify this part
| + | |
- | | + | |
- | ==8/1/2010==
| + | |
- | | + | |
- | '''Nanodrop of pBAD and GFP verification'''
| + | |
- | | + | |
- | The miniprep was performed for the two cultures started last night according the the MODIFIED miniprep protocol. A frozen stock was made of the pBAD biobrick and placed in the -80C freezer
| + | |
- | | + | |
- | The DNA concentrations reported by the nanodrop are as follows
| + | |
- | | + | |
- | *pBAD: 386 ng/uL (260/280=2.02 and 260/230=2.32)
| + | |
- | *GFP: 25.8 ng/uL (260/280=2.22 and 260/230=2.72)
| + | |
- | | + | |
- | The pBAD grew out very dense and when IPTG is added the copy number is over 100 so the DNA concentration was very high
| + | |
- | | + | |
- | '''Digest of pBAD and GFP verification'''
| + | |
| | | |
- | The parts were digested according to the protocol in the protocol section of the wiki for a 50 uL reaction volume
| + | Members of this team include [[User:annlesn|Ann Lesnefsky]], [[User:rajabiab|Bryce Rajabian]], [[User:chillywings|Marcus Lehr]], [[User:Infekt|Alex Pyden]], [[User:Jeremigo|Jeremy Golden]], [[User:Seongkyu|John Seong Kyu Yang]] and [[User:kilholee|Kilho Lee]]. |
| | | |
- | *GFP #3 was digested with XbaI and PstI
| + | ==Monthly Notebooks== |
- | *GFP #4 was digested with EcoRI and XbaI
| + | Notebooks are ordered by month. |
- | *pBAD #1 was digested with EcoRI and SpeI
| + | |
- | *pBAD #2 was digested with SpeI and PstI
| + | |
| | | |
- | ==8/2/2010==
| + | [[Team:Michigan/Oil_Sands_June_July | June/July]] |
| | | |
- | '''Gel of Digested Parts'''
| + | [[Team:Michigan/Oil_Sands_August_September | August/September]] |
| | | |
- | [[Image:7-30-2010_Biofilm_formation_in_LB_media_jpeg.jpg|500px]] | + | [[Team:Michigan/Oil_Sands_October | October]] |
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- | *Lane 1: invitrogen 1 kb plus ladder
| + | =='''In the Lab'''== |
- | *Lane 2: GFP #3 cut with XbaI and PstI (GFP: 720 bp Backbone: 2079 bp)
| + | <html> |
- | *Lane 3: GFP #4 cut with EcoRI and XbaI
| + | <iframe src="http://www-personal.umich.edu/~shanwu/wiki_photos/src/themes/classic/classic-demo.html" width="100%" height="650px" scrolling="no"></iframe> |
- | *Lane 4: uncut GFP plamid (only 1 uL left so did not show up on gel)
| + | </html> |
- | *Lane 5: pBAD cut with EcoRI and SpeI(pBAD: 1210 bp backbone: 4425 bp)
| + | |
- | *Lane 6: pBAD cut with SpeI and PstI
| + | |
- | *Lane 7: uncut pBAD (loaded very small amount into gel by accident)
| + | |
- | *Lane 8: uncut pBAD (loaded 1 uL of miniprep into gel)
| + | |