Team:Osaka/Notebook

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# Gel electrophoresis
# Gel electrophoresis
# (RESULTS?)
# (RESULTS?)
-
# Transfer to solution culture: 021, 025, 026, 034, 036
+
# 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

Revision as of 14:38, 13 October 2010

Calendar

July
Week S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
week1 25 26 27 28 29 30 31
August
Week S M T W T F S
week2 1 2 3 4 5 6 7
week3 8 9 10 11 12 13 14
week4 15 16 17 18 19 20 21
week5 22 23 24 25 26 27 28
week6 29 30 31
September
Week S M T W T F S
week6 1 2 3 4
week7 5 6 7 8 9 10 11
week8 12 13 14 15 16 17 18
week9 19 20 21 22 23 24 25
week10 26 27 28 29 30
October
Week S M T W T F S
week10 1 2
week11 3 4 5 6 7 8 9
week12 10 11 12 13 14 15 16
week13 17 18 19 20 21 22 23
week14 24 25 26 27 28 29 30
week15 31
November
Week S M T W T F S
week15 1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30










Notebook

October 1 (Fri)

  1. Gel electrophoresis (?)
  2. 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
  3. Transformation of ligation products
  4. PCR of ENO2 promoter, ADH2 promoter, SUC2 signal sequence
  5. Parts from NYU team:
IDPart NameResistanceDescription
R1<bbpart>BBa_K416003</bbpart>A,Kyeast secretion tag
R2<bbpart>BBa_K416004</bbpart>AAga2 yeast cell surface display tag with linker
  1. Miniprep of 026, 028, 030
  2. Restriction digest
    • 026, 028 with XbaI, PstI
    • 030 with EcoRI, SpeI or EcoRI only
  3. Gel electrophoresis
  4. (RESULTS?)
  5. Transfer to solution culture: 021, 025, 026, 034, 035

October 2 (Sat)

  1. Restriction digest of plasmid backbones: 1-5A, 1-3A with XbaI, PstI
  2. Electrophoresis & purification from gel
  3. 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?)
  4. PCR cloning from yeast genome ENO2, ADH2 promoters
  5. Transfer to culture solution: 006, 007, 010, 011, 036, 037, 038
  6. Miniprep of 021, 025, 026, 034, 035 followed by restriction digest with XbaI, PstI
  7. Gel electrophoresis
    • 021 - OK
    • 025 - OK
    • 026 - OK
    • 034 - bad
    • 035 - OK

October 3 (Sun)

  1. Miniprep of 006, 007, 010, 011, 036, 037, 038 followed by restriction digest with XbaI, PstI
  2. Restriction digest of yesterday's minipreps: 034, 035, 036 with EcoRI, SpeI
  3. Gel electrophoresis
    • 006 - ok
    • 007 - ?
    • 010 - bad
    • 011 - bad
    • 034 - bad
    • 035 - ok
    • 036 - ok
    • 037 - ?
    • 038 - not sufficiently digested?
  4. 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
  5. 1-2J, 1-18F, 007, 037 with EcoRI, PstI
  6. Gel electrophoresis
    • (RESULTS?)
  7. Polyacrylamide gel electrophoresis of SUC2 PCR product & elution (recipe/protocol elsewhere)
    • elution buffer added & overnight shaking incubation at 37°C
  8. Cut check of R1, R2 with EcoRI, SpeI
  9. PCR cloning from yeast genome ADH2, ENO2 again
  10. Ligation & transformation:
    • 039: 001 as upstream, 021 as downstream, 1-5A as vector
  11. Cut check of PCR products
    • results bad; repeat!
  12. PCR cloning from yeast genome: ADH2 promoter


TO CHECK/CONFIRM

  • 'K1' (mentioned on 9/250 -> where did it come from?
  • 9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
  • 9/24~27 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)

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