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Team:UIUC Illinois/Protocols
From 2014.igem.org
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<li>Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.<br> | <li>Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.<br> | ||
| - | + | <p style="text-indent:30px;">a. Pre-warm the MRS for faster growth</p><br> | |
| - | + | <p style="text-indent:30px;">b. Take 3.5-5.5 hours to get to 0.5</p><br> | |
| - | b. Take 3.5-5.5 hours to get to 0.5<br> | + | <p style="text-indent:30px;">c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker</p><br> |
| - | c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker< | + | |
| - | + | ||
</li> | </li> | ||
<li>Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB. Centrifuge in 2, 50 ml conical tubes<br> | <li>Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB. Centrifuge in 2, 50 ml conical tubes<br> | ||
| - | + | <p style="text-indent:30px;">a. Resuspend each pellet in 20 ml 3.5X SMEB (40 ml total)</p><br> | |
| - | + | <p style="text-indent:30px;">b. Can combine into one tube if desired but use 40 ml per wash</p><br> | |
| - | b. Can combine into one tube if desired but use 40 ml per wash<br> | + | </li> |
| - | + | ||
<li>Repeat step 2 at least twice for three total washes.</li><br> | <li>Repeat step 2 at least twice for three total washes.</li><br> | ||
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<li>Transfer cells (gently) to 3.0mL MRS and incubate at 37C.<br> | <li>Transfer cells (gently) to 3.0mL MRS and incubate at 37C.<br> | ||
| - | <p style="text-indent:30px;">a. ~16 hours is sufficient<br> | + | <p style="text-indent:30px;">a. ~16 hours is sufficient</p><br> |
| - | b. Does not need to be in anaerobic shaker but no shaking<br> | + | <p style="text-indent:30px;">b. Does not need to be in anaerobic shaker but no shaking</p><br> |
| - | c. We use small culture tubes but you could use falcon tubes<br> | + | <p style="text-indent:30px;">c. We use small culture tubes but you could use falcon tubes</p><br> |
| - | + | </li> | |
<li>Dilute cells accordingly and plate on MRS plus appropriate antibiotic.<br> | <li>Dilute cells accordingly and plate on MRS plus appropriate antibiotic.<br> | ||
| - | <p style="text-indent:30px;">a. L. gasseri ATCC 33323 – 5.0 ug/ml erythromycin<br> | + | <p style="text-indent:30px;">a. L. gasseri ATCC 33323 – 5.0 ug/ml erythromycin</p><br> |
| - | b. Make sure you plate a negative control<br> | + | <p style="text-indent:30px;">b. Make sure you plate a negative control</p><br> |
| - | c. I usually use add 40ml and 200ml on two plates each<br> | + | <p style="text-indent:30px;">c. I usually use add 40ml and 200ml on two plates each</p><br> |
| - | + | </li> | |
<li>Incubate 2-3 days in anaerobic chamber at 37C<br></li> | <li>Incubate 2-3 days in anaerobic chamber at 37C<br></li> | ||
Revision as of 01:33, 18 October 2014
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Gel Electrophoresis
Summarize Protocol Here
PCR Set-Up
Summary Paragraph
Bacterial Transformation
Summarize Protocol Here
Restriction Endonuclease Digest
Summary Paragraph
CaCl2 Competent Cell Protocol
Summarize Protocol Here
Genomic DNA Purification
Summary Paragraph
Resting Cell Assay
Summarize Protocol Here
Colony PCR
Summary Paragraph
Lactobacillus Transformation
- Inoculate 100mL MRS with 1mL of an overnight culture and incubate at 37C until it reaches an OD600 = 0.5 – 0.8.
a. Pre-warm the MRS for faster growth
b. Take 3.5-5.5 hours to get to 0.5
c. All liquid cultures can be done outside the anaerobic chamber; however, do not use a shaker
- Centrifuge culture at 4000rpm for 10min at 4C and wash with cold 3.5X SMEB. Centrifuge in 2, 50 ml conical tubes
a. Resuspend each pellet in 20 ml 3.5X SMEB (40 ml total)
b. Can combine into one tube if desired but use 40 ml per wash
- Repeat step 2 at least twice for three total washes.
- Resuspend cells in 1.0mL 3.5X SMEB (concentrate 100 fold).
a. Mix well using a pipettor
- Transfer 0.2mL cells to a microfuge tube and add DNA; mix and transfer to a cold 0.2cm cuvette.
- Electroporate at 2.45kV, 25μFD, 200Ω.
a. We now use an eppendorf electoporator that has only a voltage setting (we use 2.5 kV; typical time constant is ~3.3)
- Transfer cells (gently) to 3.0mL MRS and incubate at 37C.
a. ~16 hours is sufficient
b. Does not need to be in anaerobic shaker but no shaking
c. We use small culture tubes but you could use falcon tubes
- Dilute cells accordingly and plate on MRS plus appropriate antibiotic.
a. L. gasseri ATCC 33323 – 5.0 ug/ml erythromycin
b. Make sure you plate a negative control
c. I usually use add 40ml and 200ml on two plates each
- Incubate 2-3 days in anaerobic chamber at 37C
The Institute for Genomic Biology
Biosystems Design, South wing, 2nd floor
University of Illinois at Urbana-Champaign
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61801 Urbana, Illinois
illinoisigem@gmail.com
illinoisigem@gmail.com 
