Team:Arizona State/Notebook/July
From 2011.igem.org
(Difference between revisions)
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* restriction: | * restriction: | ||
:* seq1, da, ra (ES, EX) in PSB1K3 | :* seq1, da, ra (ES, EX) in PSB1K3 | ||
- | * ligation | + | * ligation: |
:* seq1 + seq1, da + da, ra + ra | :* seq1 + seq1, da + da, ra + ra | ||
:* transformation of ligation products, plated on kan (NEB cells) | :* transformation of ligation products, plated on kan (NEB cells) | ||
- | * ligation | + | * ligation: |
:* seq1 (ES, XP) + PSB1A3 (EP) | :* seq1 (ES, XP) + PSB1A3 (EP) | ||
:* transformation, plated on amp (NEB cells) | :* transformation, plated on amp (NEB cells) | ||
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* transformation results: | * transformation results: | ||
:* kan plates: | :* kan plates: | ||
- | ::* all successful (?) (seq1 + seq1, da + da, ra + ra), will be sequenced (ordered | + | ::* all successful (?) (seq1 + seq1, da + da, ra + ra), will be sequenced (ordered biobrick forward / reverse primers) |
:::* made liquid culture for miniprep tomorrow | :::* made liquid culture for miniprep tomorrow | ||
:* amp plates: | :* amp plates: | ||
::* no success | ::* no success | ||
* PCR: | * PCR: | ||
- | :* cas_b, cas_c both unsuccessful | + | :* cas_b, cas_c both unsuccessful |
:* cas_d run, will visualize tomorrow | :* cas_d run, will visualize tomorrow | ||
:* adjusting settings for cas_e_f, cas_3_r: 2 parallel | :* adjusting settings for cas_e_f, cas_3_r: 2 parallel | ||
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* Glycerol stock of DADA, RARA, Seq1Seq1 all in PSB1K3 | * Glycerol stock of DADA, RARA, Seq1Seq1 all in PSB1K3 | ||
* Gel of "homerun" attempt from yesterday, Block A touchdown + Block B constant 70 degC | * Gel of "homerun" attempt from yesterday, Block A touchdown + Block B constant 70 degC | ||
- | :* Unsuccessful, deformed gel near top, nonspecific binding, weird band at ~800bp | + | :* Unsuccessful, deformed gel near top, nonspecific binding, weird band at ~800bp |
* Met w/ 3 grad students (Kurt, Jack, Bo) and had good discussion | * Met w/ 3 grad students (Kurt, Jack, Bo) and had good discussion | ||
:* Gel techniques: doing gels in 4 degree room, using X-tracta gel removers for gel band extraction | :* Gel techniques: doing gels in 4 degree room, using X-tracta gel removers for gel band extraction | ||
- | :* Assembly: problems with large insertions | + | :* Assembly: problems with large insertions |
:* Infusion (clonetech) | :* Infusion (clonetech) | ||
:* Transformation of biobrick promoter J23101 onto amp (2 plates) | :* Transformation of biobrick promoter J23101 onto amp (2 plates) | ||
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:* DADA (ES, EX) | :* DADA (ES, EX) | ||
:* RARA (ES, EX) | :* RARA (ES, EX) | ||
- | * Ligation (protocol | + | * Ligation (protocol from Open Wetware): |
:* 2x Seq1Seq1 | :* 2x Seq1Seq1 | ||
:* 2x DADA | :* 2x DADA | ||
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:* 2x seq1, da, ra 2 ways: | :* 2x seq1, da, ra 2 ways: | ||
::* psb1k3 | ::* psb1k3 | ||
- | ::* psb1a3 | + | ::* psb1a3 |
* transformation of 14 plates: | * transformation of 14 plates: | ||
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* liquid cultures of: | * liquid cultures of: | ||
:* promoter | :* promoter | ||
- | :* e0840 | + | :* e0840 |
:* 2x seq1, da, ra | :* 2x seq1, da, ra | ||
* miniprep using new ethanol protocol: | * miniprep using new ethanol protocol: | ||
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:* polymerase problems? | :* polymerase problems? | ||
:* something w/ master mix? | :* something w/ master mix? | ||
- | :* Trinette didn't like the train | + | :* Trinette the PCR machine didn't like the train of thermocycles running on her all day |
- | + | ||
:* PCR gnomes (DNA trafficking) | :* PCR gnomes (DNA trafficking) | ||
* PCR: | * PCR: | ||
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::* final: 57 | ::* final: 57 | ||
:* gel results: | :* gel results: | ||
- | ::* ABCDE: very faint band | + | ::* ABCDE: very faint band |
::* cas3: no bands | ::* cas3: no bands | ||
== Monday, July 18 == | == Monday, July 18 == | ||
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::* gradient with 3 rows from 72 to 55 | ::* gradient with 3 rows from 72 to 55 | ||
* Late night gel results: (Madeline and Joseph) | * Late night gel results: (Madeline and Joseph) | ||
- | :* CasABCDE | + | :* CasABCDE: broad bands, perhaps desired band at ~4300 faintly @ higher magnesium concentrations |
- | :* Cas3 | + | :* Cas3: broad bands, also no ladder |
::* So we THINK we got the desired band in the "midrange" temperature, which corresponds to "third up" row and relatively high magnesium chloride concentration | ::* So we THINK we got the desired band in the "midrange" temperature, which corresponds to "third up" row and relatively high magnesium chloride concentration | ||
- | ::* | + | ::* SO we should do another PCR at approximately slightly above 60 somewhere |
::* suggestion: gradient between 65 and 60 | ::* suggestion: gradient between 65 and 60 | ||
::* also: what does it mean that it is curved? | ::* also: what does it mean that it is curved? | ||
- | ::* | + | ::* Also: what is the brightness at the bottom? happened in both gels... |
* RLT: | * RLT: | ||
* tried to gel E0840 cut w/ XP to get higher concentration of insert for ligation | * tried to gel E0840 cut w/ XP to get higher concentration of insert for ligation | ||
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:* GOT ICE ACCESS!!! | :* GOT ICE ACCESS!!! | ||
:* Kylie/Keith battle has begun | :* Kylie/Keith battle has begun | ||
- | :* Xiao is proud of us | + | :* Xiao is proud of us |
* To do: | * To do: | ||
:* email iGEM to see what's up w/ Indianapolis accommodations | :* email iGEM to see what's up w/ Indianapolis accommodations | ||
:* fill out form | :* fill out form | ||
- | |||
== Wednesday, July 20 == | == Wednesday, July 20 == | ||
* transformation results: | * transformation results: | ||
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:* E0840 | :* E0840 | ||
:* Seq1, RA, DA | :* Seq1, RA, DA | ||
- | * Gel of RE results | + | * Gel of RE results |
:* Very clear bright bands for (ES) E0840 vector, insert | :* Very clear bright bands for (ES) E0840 vector, insert | ||
::* Extracted insert | ::* Extracted insert | ||
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* PCR Insert Amplification, Attempt #1 | * PCR Insert Amplification, Attempt #1 | ||
:* Taq polymerase from Bioline | :* Taq polymerase from Bioline | ||
- | |||
:* Notable: 30 sec elongation time | :* Notable: 30 sec elongation time | ||
- | * Gel of PCR Results | + | * Gel of PCR Results |
:* faint bands at ~1000bp | :* faint bands at ~1000bp | ||
:* likely due to 30sec elongation, will try again w/shorter time | :* likely due to 30sec elongation, will try again w/shorter time | ||
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* PCR Insert Amplification, Attempt #2 | * PCR Insert Amplification, Attempt #2 | ||
:* Used Phusion instead of Biolase product | :* Used Phusion instead of Biolase product | ||
- | |||
:* Annealing temp: 58 deg, based off of NEB calculator as recommended by Phusion protocol | :* Annealing temp: 58 deg, based off of NEB calculator as recommended by Phusion protocol | ||
:* Annealing time: 10 sec (low end) | :* Annealing time: 10 sec (low end) | ||
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:* 25 total cycles (low end) | :* 25 total cycles (low end) | ||
:* 5 minute extension (low end) | :* 5 minute extension (low end) | ||
- | * Gel Result | + | * Gel Result: |
:* Very strong, medium thick bands | :* Very strong, medium thick bands | ||
:* Seq1 ~ 130-180bp | :* Seq1 ~ 130-180bp | ||
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* PCR Amplification of pUC57 inserts | * PCR Amplification of pUC57 inserts | ||
:* 4 second elongation (instead of 5 sec), 35 cycles (instead of 25) | :* 4 second elongation (instead of 5 sec), 35 cycles (instead of 25) | ||
- | * Gel Results | + | * Gel Results |
:* same major bands as first attempt (~150 for seq1, ~200 for DA, RA) | :* same major bands as first attempt (~150 for seq1, ~200 for DA, RA) | ||
:* more minor bands than before | :* more minor bands than before | ||
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:* Seq1, DA, RA 1x in psb1A3 (Forward/Reverse) | :* Seq1, DA, RA 1x in psb1A3 (Forward/Reverse) | ||
== Wednesday, July 27 == | == Wednesday, July 27 == | ||
- | * ligate 2x in psb1k3 | + | * ligate 2x in psb1k3 |
:* using overnight digests | :* using overnight digests | ||
::* Seq1 in pSB1A3 (ES, XP) | ::* Seq1 in pSB1A3 (ES, XP) | ||
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* PCR: | * PCR: | ||
:* casABCDE with new and old primers and a temp gradient for both | :* casABCDE with new and old primers and a temp gradient for both | ||
- | :* new: | + | :* new: 64->69, three rows of 3 tubes |
- | :* old: | + | :* old: 63->66, three rows of 3 tubes |
* Gel results: | * Gel results: | ||
:* new -- no good | :* new -- no good | ||
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* meeting with grad students: | * meeting with grad students: | ||
:* homology between casA and human immune proteins (which ones?) | :* homology between casA and human immune proteins (which ones?) | ||
- | ::* could be an evolutionary | + | ::* could be an evolutionary thing |
:* look into this could also be | :* look into this could also be | ||
::* conserved functional domains | ::* conserved functional domains |
Revision as of 07:11, 28 September 2011
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