Team:Caltech/Week 7

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Latest revision as of 01:05, 4 August 2010

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Monday 7/26

CPCR gel featuring L16, L19, L23, L24, L31 & L34. (7/26)

CPCR gel featuring L34, L35, L40, L42, L43 & L44. (7/26)

Started PCR reaction to extract the HSP (K112400) from its Berkeley Standard backbone (BBb). (Used same PCR parameters as CPCR from 7/22.
Group meeting!
- By next week: Need to design test-construct experiments, as they will be ready sometime early next week.
  - Can use PCR machine to achieve easy temperature gradients
  - Use spectrophotometer and eventually microscope to assay fluorescence.
  - Consider a plate assay?
- AIBN issues:
  - Consider using low-concentration DMSO to help dissolve AIBN in LB/cell mixture.
  - Need to figure out how much unreacted (or otherwise) AIBN remains after mixing with cells.
  - Need to test crosslinking using hydrolyzed and epoxidized oil mixed with lysate and with cells to tweak concentration of AIBN needed, and to see what it makes.
- TGase:
  - purchase urea?
  - To prevent a solid plug forming upon addition of TGase powder to sample, prepare liquid stock of concentrated TGase.
- Wiki:
  - Should visit biofab.org for research material for human impact segment.
- In general, we should set goals for both the end of SURF and for the end of the summer and determine what we need to do to get on track for them.
  - Printing in 2D
  - Working AND gate, at least in bulk fluid
  - Characterization of 3D hydrogel/plastic
  - Crosslinking data
PCR purified K112400 PCR product & digested as plasmid insert.
Transformed RFP bricks of verious resistances to use as backbone for future ligations
Ran 2 gels of today's CPCR products.

Tuesday 7/27

CPCR gel featuring L16, L19, L31, L34, & L35. (7/22)

Began more CPCR on failed ones from yesterday: L16, L19, L31, L34, L35
Began more digests of miniprepped DNA from K112400
Made more electrocompetent cells. Began two test transformations.
Transformed ligation products: L42, L46, L47 and the HSP.
Began 11 new ligations:
- L48: R0082 + I13504 + Cm
- L31: L17 + K215000 + Kan
- L34: L17 + L21 + Amp
- L35: L18 + L21 + Amp
- L22: K156012 + B0034 + Cm
- L24: K156014 + B0015 + Cm
- L20: C0077 + B0015 + Cm
- L49: K112400 + L40 + Cm
- L25: B0015 + M30109 + Tet
- L26: R0077 + K124017 + Cm
- L50: L18 + L27 + Amp
Note: Need to upstream-digest more of: J23119, L17, K156014, B0015 for future ligations.

Wednesday 7/28

Began CPCR reactions on the HSP, L42, L46, L48.
Transformed all ligations from yesterday
Re-ran gel from yesterday
- Same results: looks like 19-4 is a success
Ran gel of today's CPCR to determine if the HSP ligations worked.
- All bands appear to have a 100-400bp smear and no other bands, making the results indeterminate.

Thursday 7/29

Re-did the following ligations: L16, L31, L34, L35, HSP (in pSB1C3, for submission), L42, L46, L47
Started epic amounts of colony PCR:
- 3 colonies of each: L42, L46, L31, HSP-Amp, L35, L50, L34, L48, L24, L49, L22, L20
- Gels for each are included to the right ->
- Successful: L42-4/-5, L46-4/-5, L47-4, HSP-Amp-5/-6

Friday 7/30

Prepped 47-4 (HSP + B0034), L42-4/-5, L46-4/-5 (HSP + R0077), HSP-Amp-5/-6
Began a ton of digests:
- 2x Tet backbone
- U19: K156012 (us)
- U20: K156014 (us)
- U21: C0077 (us)
- U22: L19 (us)
- U23: L46 (us)
- U24: L47 (us)
- U25: L17 (us)
- D17: B0034 (ds)
- D18: B0015 (ds)
- D19: L18 (ds)
- D20: L19 (ds)
- D21: HSP-Amp (ds)

Weekend 7/31-8/1

Saturday 7/31

Transformed L22, L24, L33, L43
- Failed: L51, L52, L53, L25 (cuvette arced)
Ran gel of M30109 CPCR product: no bands!
- Is M30109 incorrect?

Sunday 8/1

Removed transformation plates: L22 & L24 were successful; L33 & L53 failed.

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@@ Line 4: / Line 4: @@
 ==Monday 7/26==
+[[Image:CIT-cpcr-gel1-7-26.jpg|200px|thumb|CPCR gel featuring L16, L19, L23, L24, L31 & L34. (7/26)]]
+[[Image:CIT-cpcr-gel2-7-26.jpg|200px|thumb|CPCR gel featuring L34, L35, L40, L42, L43 & L44. (7/26)]]
+* Started PCR reaction to extract the HSP (K112400) from its Berkeley Standard backbone (BBb). (Used same PCR parameters as CPCR from 7/22.
+* Group meeting!
+** By next week: Need to design test-construct experiments, as they will be ready sometime early next week.
+*** Can use PCR machine to achieve easy temperature gradients
+*** Use spectrophotometer and eventually microscope to assay fluorescence.
+*** Consider a plate assay?
+** AIBN issues:
+*** Consider using low-concentration DMSO to help dissolve AIBN in LB/cell mixture.
+*** Need to figure out how much unreacted (or otherwise) AIBN remains after mixing with cells.
+*** Need to test crosslinking using hydrolyzed and epoxidized oil mixed with lysate and with cells to tweak concentration of AIBN needed, and to see what it makes.
+** TGase:
+*** purchase urea?
+*** To prevent a solid plug forming upon addition of TGase powder to sample, prepare liquid stock of concentrated TGase.
+** Wiki:
+*** Should visit biofab.org for research material for human impact segment.
+** In general, we should set goals for both the end of SURF and for the end of the summer and determine what we need to do to get on track for them.
+*** Printing in 2D
+*** Working AND gate, at least in bulk fluid
+*** Characterization of 3D hydrogel/plastic
+*** Crosslinking data
+* PCR purified K112400 PCR product & digested as plasmid insert.
+* Transformed RFP bricks of verious resistances to use as backbone for future ligations
+* Ran 2 gels of today's CPCR products.
 ==Tuesday 7/27==
+[[Image:CIT-cpcr-gel-7-27.jpg|200px|thumb|CPCR gel featuring L16, L19, L31, L34, & L35. (7/22)]]
+* Began more CPCR on failed ones from yesterday: L16, L19, L31, L34, L35
+* Began more digests of miniprepped DNA from K112400
+* Made more electrocompetent cells. Began two test transformations.
+* Transformed ligation products: L42, L46, L47 and the HSP.
+* Began 11 new ligations:
+** L48: R0082 + I13504 + Cm
+** L31: L17 + K215000 + Kan
+** L34: L17 + L21 + Amp
+** L35: L18 + L21 + Amp
+** L22: K156012 + B0034 + Cm
+** L24: K156014 + B0015 + Cm
+** L20: C0077 + B0015 + Cm
+** L49: K112400 + L40 + Cm
+** L25: B0015 + M30109 + Tet
+** L26: R0077 + K124017 + Cm
+** L50: L18 + L27 + Amp
+* Note: Need to upstream-digest more of: J23119, L17, K156014, B0015 for future ligations.
 ==Wednesday 7/28==
+* Began CPCR reactions on the HSP, L42, L46, L48.
+* Transformed all ligations from yesterday
+* Re-ran gel from yesterday
+** Same results: looks like 19-4 is a success
+* Ran gel of today's CPCR to determine if the HSP ligations worked.
+** All bands appear to have a 100-400bp smear and no other bands, making the results indeterminate.
 ==Thursday 7/29==
+* Re-did the following ligations: L16, L31, L34, L35, HSP (in pSB1C3, for submission), L42, L46, L47
+* Started epic amounts of colony PCR:
+** 3 colonies of each: L42, L46, L31, HSP-Amp, L35, L50, L34, L48, L24, L49, L22, L20
+** Gels for each are included to the right ->
+** Successful: L42-4/-5, L46-4/-5, L47-4, HSP-Amp-5/-6
 ==Friday 7/30==
+* Prepped 47-4 (HSP + B0034), L42-4/-5, L46-4/-5 (HSP + R0077), HSP-Amp-5/-6
+* Began a ton of digests:
+** 2x Tet backbone
+** U19: K156012 (us)
+** U20: K156014 (us)
+** U21: C0077 (us)
+** U22: L19 (us)
+** U23: L46 (us)
+** U24: L47 (us)
+** U25: L17 (us)
+** D17: B0034 (ds)
+** D18: B0015 (ds)
+** D19: L18 (ds)
+** D20: L19 (ds)
+** D21: HSP-Amp (ds)
 ==Weekend 7/31-8/1==
+=== Saturday 7/31 ===
+* Transformed L22, L24, L33, L43
+** Failed: L51, L52, L53, L25 (cuvette arced)
+* Ran gel of M30109 CPCR product: no bands!
+** Is M30109 incorrect?
+=== Sunday 8/1 ===
+* Removed transformation plates: L22 & L24 were successful; L33 & L53 failed.
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