Team:Osaka/week10

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Latest revision as of 08:00, 18 October 2010

September 26 (Sun)

Transfer to culture solution (yesterday's transformations)
Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
Restriction digest
- 1-6N, 1-6I with EcoRI, SpeI
- 2-2F, 021 with XbaI, PstI
Gel electrophoresis
- (RESULTS?)
Miniprep of parts moved to solution culture this morning
Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
- Cel8 - ok
- Cel44 - ??
- Man26 - ok
- Xyn10 - ??
Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
- 026 - ADH1 terminator
- 027 - ADH2 promoter
- 028 - CYC1 terminator
- 029 - ENO2 promoter
- 030 - SUC2 leader sequence
- 031 - glr (glutamate racemase)
Transformation of ligation products
Transfer of yesterday's transformations to solution culture: 001-2, 025

September 27 (Mon)

PCR of Man26, CelB
PCR of Man48 (??), CBM from XynAcc
Restriction digests
- 1-2M with EcoRI, SpeI for assembly later
- Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (for checking?)
- 001-2 with EcoRI, SpeI
- 025 with XbaI, PstI
Gel electrophoresis
Ligations
- 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
- 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
- 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (remake using 001-2)
- 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (" ")
Transformation of ligation products
Transfer to solution culture: 026, 027, 028, 029, 030, 031

September 28 (Tue)

Miniprep of 026, 027, 028, 030, 031
Restriction digest
- 026, 028, 031 with XbaI, PstI
- 027, 030 with EcoRI, SpeI
Gel electrophoresis
- (RESULTS?)
Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
Transformation of ligation products as well as 3-2P

ID	Part Name	Resistance	Description
3-2P	<bbpart>BBa_</bbpart>	A	??

September 29 (Wed)

Transfer to solution culture 025, 026, 027, 028, 030, 031
Ligations (repeat of 9/26 with different dilution of 1-1C)
- 026 - ADH1 terminator
- 027 - ADH2 promoter
- 028 - CYC1 terminator
- 029 - ENO2 promoter
- 030 - SUC2 leader sequence
- 031 - glr (glutamate racemase)
Transformation of 025, 026, 027, 028, 030, 031 (repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?)

PCR
- pgsB - cloning from plasmid
- pgsB - amplification from 021
- Man28 - amplification from previous product
PCR purification of products

PCR of CelB, XynA-CBM

Miniprep of cultures inoculated this morning (total culture time: 12hr)
- 025 -> ok (colorless)
- 027 -> turned red; discarded
- others: ok?
Restriction digest of 026, 028, 031 with XbaI, PstI
Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
Gel electrophoresis of PCR products

Restriction digests:
- pgsB, Man (??) with EcoRI, SpeI
- today's miniprepped plasmids with EcoRI
- 1-1C with EcoRI, SpeI

September 30 (Thu)

PCR cloning
- CM10
- CelB (repeat)
- ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
- glr (repeat)
PCR purification: CM10, XynA-CBM
Restriction digest of all above PCR products with EcoRI, SpeI
Gel electrophoresis

Ligation of PCR products to 1-1C backbone
- 021: pgsB
- 025: XynA-CBM
- 026: ADH1 terminator
- 028: CYC1 terminator
- 031: glr
- 034: Man
- 035: CM10
Transformation of ligation products

PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
Gel electrophoresis
- (RESULTS?)

Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
Restriction digest of miniprepped parts
- 004, 005, 032 with EcoRI, SpeI (same as earlier today)
- 033, 3-2P (same as on 9/29)
Gel electrophoresis
- (RESULTS?)

October 1 (Fri)

Gel electrophoresis (?)
Ligations
- 006: 004 as upstream, FcenA as downstream, 1-5A as vector
- 007: 005 as upstream, FcenA as downstream, 1-5A as vector
- 010: 004 as upstream, Fcex as downstream, 1-5A as vector
- 011: 005 as upstream, Fcex as downstream, 1-5A as vector
- 036: 032 as upstream, 033 as downstream, 1-5A as vector
- 037: 004 as upstream, 024 as downstream, 1-1C as vector
- 038: 005 as upstream, 024 as downstream, 1-1C as vector
Transformation of ligation products
PCR of ENO2 promoter, ADH2 promoter, SUC2 signal sequence
Parts from NYU team:

ID	Part Name	Resistance	Description
R1	<bbpart>BBa_K416003</bbpart>	A,K	yeast secretion tag
R2	<bbpart>BBa_K416004</bbpart>	A	Aga2 yeast cell surface display tag with linker

Miniprep of 026, 028, 030
Restriction digest
- 026, 028 with XbaI, PstI
- 030 with EcoRI, SpeI or EcoRI only
Gel electrophoresis
(RESULTS?)
Transfer to solution culture: 021, 025, 026, 034, 035

October 2 (Sat)

Restriction digest of plasmid backbones: 1-5A, 1-3A with XbaI, PstI
Electrophoresis & purification from gel
PCR of SUC2 signal sequence
- primers diluted with MiliQ water from 100μM to 5μM
- Taq polymerase added before starting (as opposed to after initial denaturation step)
- gel run to check -> (RESULTS?)
PCR cloning from yeast genome ENO2, ADH2 promoters
Transfer to culture solution: 006, 007, 010, 011, 036, 037, 038
Miniprep of 021, 025, 026, 034, 035 followed by restriction digest with XbaI, PstI
Gel electrophoresis
- 021 - OK
- 025 - OK
- 026 - OK
- 034 - bad
- 035 - OK

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@@ Line 164: / Line 164: @@
 #* 035 - OK
-==October 3 (Sun)==
-# Miniprep of 006, 007, 010, 011, 036, 037, 038 followed by restriction digest with XbaI, PstI
-# Restriction digest of yesterday's minipreps: 034, 035, 036 with EcoRI, SpeI
-# Gel electrophoresis
-#* 006 - ok
-#* 007 - ?
-#* 010 - bad
-#* 011 - bad
-#* 034 - bad
-#* 035 - ok
-#* 036 - ok
-#* 037 - ?
-#* 038 - not sufficiently digested?
-# Another round of restriction digest:
-#* spare samples of 010, 011, 037 with XbaI, PstI as above (''2 colonies were cultured in solution & miniprepped'')
-#* 038: more XbaI, PstI added to previous tube
-# 1-2J, 1-18F, 007, 037 with EcoRI, PstI
-# Gel electrophoresis
-#* (RESULTS?)
-# Polyacrylamide gel electrophoresis of SUC2 PCR product & elution (recipe/protocol elsewhere)
-#* elution buffer added & overnight shaking incubation at 37°C
-# Cut check of R1, R2 with EcoRI, SpeI
-# PCR cloning from yeast genome ADH2, ENO2 ''again''
-# Ligation & transformation:
-#* 039: 001 as upstream, 021 as downstream, 1-5A as vector
-# Cut check of PCR products
-#* ''results bad; repeat!''
-# PCR cloning from yeast genome: ADH2 promoter
-==October 4 (Mon)==
-# Gel electrophoresis
-#* sample?
-# Transformation og new genes arrived from Utha University.
-#* HlyA  pSB3K3  19.3mg/ml
-#* ToRA  pSB3K3  12.9mg/l
-#* GeneIII  pSB1AK3  197.7mg/ml
-# Transfer to soltion culture: 039.
-# Restriction digest of 007 with <i>Eco</i>RI.
-# Gel electrophoresis: 007.
-# Restriction digest of eluted SUC with <i>Eco</i>RI and <i>Spe</i>I.
-# Ligation
-#* 008(K): 001(upstream), 2-20H(downstream), 1-5A(vector)
-#* 009(K): 001(upstream), 2-20J(downstream), 1-5A(vector)
-#* 010(K): 004(upstream), Fcex(023, downstream), 1-5A(vector)
-#* 015(C): 001(upstream), 1-13D(downstream), 1-3A(vector)
-#* 016(C): 007(upstream), 1-13D(downstream), 1-3A(vector)
-#* 044(C): 001-2(upstream), F1(downstream), 1-3A(vector)
-#* 045(A): 005(upstream), 024(beta-gluctosidase, downstream), 1-1C(vector)
-#* 046(A): 004(upstream), 024(downstream), 1-1C(vector)
-==October 5 (tue)==
-# PCR of glf, Man, ADH2, ENO2, and Gel electrophoresis.
-# Transformation of yesterday's ligation products.
-# PCR of glf.
-#* second time?
-# Miniprep of 039, 2-4A, 3-11l, R1.
-# Restriction digest of R1 with <i>Xba</i>I, <i>Pst</i>I, and Man, 039, 2-4A, 3-11l  with <i>Eco</i>RI, <i>Spe</i>I.
-# Gel electrophoresis of digested plasmids.
-#* R1  OK
-#* 039  OK
-#* 2-4A  OK
-#* 3-11l  X
-#* Man  OK
-#* glr  X
-# Transfer transformants to solution culture: 1-3A, 1-5A, 1-1C, HlyA, ToRA, GeneIII.
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