Team:Montreal/Notebook
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==Lab Progress== | ==Lab Progress== | ||
- | <p>'''May 21st, 2008''': <ul> | + | <p>'''May 21st, 2008''': <ul>Restriction enzyme digest was done on the J-brick with EcoRI. Gel was run on the J-brick after the restriction digest. No DNA was detected on the gel (the ladder was visible on the gel).</ul></p> |
- | <p>'''May 22nd, 2008''': <ul>Prepared TOP10 | + | <p>'''May 22nd, 2008''': <ul>Prepared TOP10 competent cells for eventual transformation.</ul> |
<ul>Performed Mini-prep on Reporter+ Cells</ul> | <ul>Performed Mini-prep on Reporter+ Cells</ul> | ||
<ul>Performed Digest and Gel on Reporter plasmid extract -- no DNA present, suggest follow-up maxi-prep</ul></p> | <ul>Performed Digest and Gel on Reporter plasmid extract -- no DNA present, suggest follow-up maxi-prep</ul></p> | ||
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<p>'''May 25th, 2008''': <ul>Diluted Reporter cells 1/1000 with 5ul Kan/mL culture for 16h incubation at 5:30pm. To be used for Maxiprep at 9:30am-1:30pm.</ul></p> | <p>'''May 25th, 2008''': <ul>Diluted Reporter cells 1/1000 with 5ul Kan/mL culture for 16h incubation at 5:30pm. To be used for Maxiprep at 9:30am-1:30pm.</ul></p> | ||
- | <p>'''May 28th, 2008''':<ul> Ran gel on Elowitz | + | <p>'''May 28th, 2008''':<ul> Ran gel on Elowitz Reporter DNA cut with EcoR1; 2 bands</ul> |
<ul>0.7 kb and 2.0 kb, confirms identity of reporter DNA.</ul> | <ul>0.7 kb and 2.0 kb, confirms identity of reporter DNA.</ul> | ||
<ul>Seeded syn-I and J-40001 into amp/kan LB and kan LB.</ul></p> | <ul>Seeded syn-I and J-40001 into amp/kan LB and kan LB.</ul></p> |
Revision as of 23:42, 16 June 2008
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Lab Protocols
Lab Progress
May 21st, 2008:
- Restriction enzyme digest was done on the J-brick with EcoRI. Gel was run on the J-brick after the restriction digest. No DNA was detected on the gel (the ladder was visible on the gel).
May 22nd, 2008:
- Prepared TOP10 competent cells for eventual transformation.
- Performed Mini-prep on Reporter+ Cells
- Performed Digest and Gel on Reporter plasmid extract -- no DNA present, suggest follow-up maxi-prep
May 23rd, 2008:
- Transformed Top10 cells with Puc19 to ensure that the competent cell procedure was successful. Growth was observed, therefore procedure was successful.
May 25th, 2008:
- Diluted Reporter cells 1/1000 with 5ul Kan/mL culture for 16h incubation at 5:30pm. To be used for Maxiprep at 9:30am-1:30pm.
May 28th, 2008:
- Ran gel on Elowitz Reporter DNA cut with EcoR1; 2 bands
- 0.7 kb and 2.0 kb, confirms identity of reporter DNA.
- Seeded syn-I and J-40001 into amp/kan LB and kan LB.
May 29th, 2008:
- Growth of J-brick in culture - No growth of I-brick on culture
- Seeded J-brick for Midi-Prep in 40mL LB with ampicillin
- Transformed TOP10 cells with both I brick and Reporter Plasmid
June 2nd, 2008:
- Growth of I-brick on culture
- Midi-prep of both I and J brick followed by gel
- Gel indicates no presence of DNA, will be confirmed by spectrophotometric assay
June 3rd, 2008:
- Seeding of 5mL cultures of both I and J brick
- Identity of colonies on I brick plates is suspect, must ensure that eventual DNA gel confirms exact restriction digest
June 4th, 2008:
- Midiprep was done on the I and the J-brick. Once the isopropanol was added, the J-brick midiprep looked clear (no DNA was eluted). DNA gel needs to be done to confirm presence of DNA in both cases.
June 9th, 2008:
- Seeded J and I-brick re-seeded for Maxi-Prep
- Gel failed to confirm presence of previously collected DNA samples of J and I-brick, will be repeated following Maxi-Prep.
June 10th, 2008:
- Since no growth was observed in the I-brick culture, the I brick was re-seeded.
- The J-brick was diluted in 500-ml of LB broth(for a Maxiprep to be done the next day).
June 11th, 2008:
- Maxiprep of the J-brick.
- Restriction digest of J-brick.
- Dilution of I-brick in 500-ml of LB broth.
June 12th, 2008:
- Maxiprep of the I-brick.
- Restriction digest and gel of June 11th and June 12th I-brick and J-brick DNA using EcoR1. Bands revealed at roughly 4000bp and 2500bp for I-brick (expected 2652bp and 3939bp). J brick single band that was not informative, new digestion to be completed tomorrow.
June 16th, 2008:
- I brick was seeded and diluted over last two days, but there was insufficient growth so it will be left to grow one more day before performing another midi-prep. This is to compliment the already successful Maxi-Prep that gave low concentrations of DNA.
- Another gel was performed of previous J-brick preps that confirmed the absence of the desired plasmid, no DNA was detected when digested with EcoR1.
- J-brick was re-transformed into TOP10 chemically competent cells and then plated on Amp+ plates.<ul/></p>