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Team:British Columbia/Notebook QS
From 2010.igem.org
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<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p> | <p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p> | ||
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p> | <p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p> | ||
| - | <p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p> | + | <p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p> |
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p> | <p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p> | ||
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p> | <p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p> | ||
Revision as of 06:32, 27 October 2010
Lessons Learned
1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer.
2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert.
3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.
4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.
5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.
6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes.
7. Check antibiotics before using them.
8. DNA is incredibly stable. DNA stocks will last a long time when stored probably, even in water.
9. Don't be afraid of asking for help or asking to use other lab groups' equipment.
10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA.
11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.
OpenWetWare (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.
See our UBC OWW notebook.
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