|
|
| Line 323: |
Line 323: |
| | ==Notebook== | | ==Notebook== |
| | | | |
| - | ===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
| |
| - |
| |
| - | ===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)
| |
| | | | |
| | ===TO CHECK/CONFIRM=== | | ===TO CHECK/CONFIRM=== |
"
