Team:UT-Tokyo/LabNote

From 2011.igem.org

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=Lab Diary=
=Lab Diary=
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For convenience sake, each part (gene, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor).
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For convenience sake, each part (genes, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor).
By pointing the mouse on the part number in the construct, you can find out the details of the part.
By pointing the mouse on the part number in the construct, you can find out the details of the part.
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Revision as of 10:40, 14 September 2011

Parts List

Table 1. List of iGEM parts
Number Part Content Plasmid Length (bp)
#1 J23119 c.Promoter (Strong) pSB1A2 35
#2 J23118 c.Promoter (Medium) BBa_J61002 35
#3 B0032 RBS pSB1A2 13
#5 B0014 d.Ter pSB1AK3 95
#9 E0240 RBS-GFP-d.Ter pSB1A2 876
#10 E0040 GFP pSB1A2 681
#11 E1010 RFP pSB2K3 723
#14 I712019 fLuc pSB1AK8 1653
#17 J52008 rLuc pSB1AK3 936
#20 R0011 lacP pSB1A2 55
#21 C0012 LacI pSB1A2 1128
#22 I712074 pT7 pSB1AK8 46
#23 K145001 T7 RNA Pol. pSB1A2 2655
#24 J22106 recAp pSB1A2 192
#27 C0083 AspA pSB2K3 1518
#28 K112808 T4 phage lysis device (no promoter) pSB1A2 1785
#29 - CheZ pSB1AK3 728
#30 K117000 Lysis gene pSB1A2 144
#31 - LexA pSB1AK3 750
#33 - sulAp pSB1AK3 67
#34 - uvrAp pSB1AK3 96
#35 - recNp
#36 R0051 cI-repressed promoter pSB1A2 49
#37 C0051 cI repressor (LVA tagged) pSB1A2 \
#38 - RecA

Lab Diary

For convenience sake, each part (genes, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor). By pointing the mouse on the part number in the construct, you can find out the details of the part.

'11/9/12 (Mon)

  • Colony PCR
    • #20-#3-14-5-2-3-17-5
    • #20-3-14-5-#2-3-17-5
    • #1-#3-14-5-2-3-17-5
    • #36-3-14-5-2-3-17-5-#20-3-37-5 (no colony)
    • #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 (PCR failed?)

Note: #20-3-37-5 colonies on the master-plate are RED. Maybe #2-3-11-5...
Note: #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...

  • PCR amplification
    • #20-3-37-5 from Master plate
    • #36-3-14-5-2-3-17-5 from 0911 MP product

Note: Only one #20-3-37-5 colony is NOT RED.

  • Miniprep.
    • #20
    • #29 (pSB1AK3)
    • #2-3-17-5
    • #2-3-37-5
    • BBa_J04450 (pSB1C3)
  • Digest
    • #36-3-14-5-2-3-17-5_ES (from PCR)
    • #20-3-37-5_XP (from PCR)
    • pSB1A3_EP
    • pSB1C3_EP
    • #20-3-37-5_XP (from MP) -> O/N
    • J04450_EP -> O/N
    • #2-9_XP -> O/N
    • #20-3-29-5_ES -> O/N
  • Gel extraction
    • #20_SP
    • #36-3-14-5_SP
    • #2-3-17-5_EX (from O/N digests)
    • #36-3-14-5-2-3-17-5_ES
    • #20-3-37-5_XP (from today's PCR digests)
  • Culture for Miniprep.
    • #20-3-37-5 from 0911 master plate -> O/N
  • Dual Luciferase Assay
    • #20-3-14-5-2-3-17-5
    • #1-3-14-5-2-3-17-5
    • #36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3
  • Ligation -> O/N
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP
    • #3-27-5_XP-#20_SP
    • #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP


'11/9/13 (Tue)

  • Colony PCR
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP (failed)
    • #3-27-5_XP-#20_SP (failed)
    • #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
  • Culture for Luciferase Assay
    • #36-3-14-5-2-3-17-5-20-3-37-5(pSB1A3, pSB1C3) (IPTG 0, 1, 10, 38, 100uM)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)

Note: Seemes OD600 should NOT exceed 0.4~0.5, or intracellular luciferase will be saturated.

  • Miniprep.
    • #20-3-14-5-2-3-17-5
    • #1-3-14-5-2-3-17-5
    • #20-3-37-5 (from a non-red colony on the master plate)
  • Gel ext.
    • #20-3-37-5_XP (from non-red colony)
    • #20-3-29-5_ES(failed)
    • #2-9_XP, pSB1C3_EP

Note: #20-3-29-5 was not cut!

  • Dual Luciferase Assay
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
    • #36-3-14-5-2-3-17-5-20-3-37-5 (#2-3-11-5?) (IPTG 0, 1, 10, 38, 100uM)
  • Sequencing preparation
    • #20-3-37-5 (from a non-red colony on the master plate)
    • #20-3-29-5
    • #3-27-5
    • #31
  • Digest
    • #20_E
    • #3-29-5_S (from MP products) (cut check)
  • Ligation
    • #20_SP-#3-27-5_XP
  • Transformation -> O/N
    • #3-14-5-2-3-17-5
    • #1-3-14-5-2-3-17-5
    • #20-3-14-5-2-3-17-5 (for plate stock)
    • #20-3-37-5 (from a non-red colony on the master plate) (for plasmid check)

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