Team:UT-Tokyo/LabNote

From 2011.igem.org

(Difference between revisions)
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=='11/9/12 (Mon)==
=='11/9/12 (Mon)==
*Colony PCR
*Colony PCR
-
**#20-#3-14-5-2-3-17-5
+
**<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-14-5-2-3-17-5
-
**#20-3-14-5-#2-3-17-5
+
**<nowiki>#</nowiki>20-3-14-5-<nowiki>#</nowiki>2-3-17-5
-
**#1-#3-14-5-2-3-17-5
+
**<nowiki>#</nowiki>1-<nowiki>#</nowiki>3-14-5-2-3-17-5
-
**#36-3-14-5-2-3-17-5-#20-3-37-5 (no colony)
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5-<nowiki>#</nowiki>20-3-37-5 (no colony)
-
**#36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 (PCR failed?)
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5-<nowiki>#</nowiki>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: <nowiki>#</nowiki>20-3-37-5 colonies on the master-plate are RED. Maybe <nowiki>#</nowiki>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...
+
Note: <nowiki>#</nowiki>36-3-14-5-2-3-17-5-<nowiki>#</nowiki>20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...
*PCR amplification
*PCR amplification
-
**#20-3-37-5 from Master plate~
+
**<nowiki>#</nowiki>20-3-37-5 from Master plate~
-
Note: Only one #20-3-37-5 colony is NOT RED.
+
Note: Only one <nowiki>#</nowiki>20-3-37-5 colony is NOT RED.
-
**#36-3-14-5-2-3-17-5 from 0911 MP product
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5 from 0911 MP product
*Miniprep.
*Miniprep.
-
**#20
+
**<nowiki>#</nowiki>20
-
**#29 (pSB1AK3)
+
**<nowiki>#</nowiki>29 (pSB1AK3)
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**#2-3-17-5
+
**<nowiki>#</nowiki>2-3-17-5
-
**#2-3-37-5
+
**<nowiki>#</nowiki>2-3-37-5
**BBa_J04450 (pSB1C3)
**BBa_J04450 (pSB1C3)
*Digest
*Digest
-
**#36-3-14-5-2-3-17-5_ES (from PCR)
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES (from PCR)
-
**#20-3-37-5_XP (from PCR)
+
**<nowiki>#</nowiki>20-3-37-5_XP (from PCR)
**pSB1A3_EP
**pSB1A3_EP
**pSB1C3_EP
**pSB1C3_EP
-
**#20-3-37-5_XP (from MP) -> ''O/N''
+
**<nowiki>#</nowiki>20-3-37-5_XP (from MP) -> ''O/N''
**J04450_EP -> ''O/N''
**J04450_EP -> ''O/N''
-
**#2-9_XP -> ''O/N''
+
**<nowiki>#</nowiki>2-9_XP -> ''O/N''
-
**#20-3-29-5_ES -> ''O/N''
+
**<nowiki>#</nowiki>20-3-29-5_ES -> ''O/N''
*Gel extraction
*Gel extraction
-
**#20_SP
+
**<nowiki>#</nowiki>20_SP
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**#36-3-14-5_SP
+
**<nowiki>#</nowiki>36-3-14-5_SP
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**#2-3-17-5_EX (from O/N digests)
+
**<nowiki>#</nowiki>2-3-17-5_EX (from O/N digests)
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**#36-3-14-5-2-3-17-5_ES
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES
-
**#20-3-37-5_XP (from today's PCR digests)
+
**<nowiki>#</nowiki>20-3-37-5_XP (from today's PCR digests)
*Culture for Miniprep.
*Culture for Miniprep.
-
**#20-3-37-5 from 0911 master plate -> ''O/N''
+
**<nowiki>#</nowiki>20-3-37-5 from 0911 master plate -> ''O/N''
*Dual Luciferase Assay
*Dual Luciferase Assay
-
**#20-3-14-5-2-3-17-5
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**<nowiki>#</nowiki>20-3-14-5-2-3-17-5
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**#1-3-14-5-2-3-17-5
+
**<nowiki>#</nowiki>1-3-14-5-2-3-17-5
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**#36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3  
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3  
*Ligation -> ''O/N''
*Ligation -> ''O/N''
-
**#36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1A3_EP
-
**#36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1C3_EP
-
**#3-27-5_XP-#20_SP
+
**<nowiki>#</nowiki>3-27-5_XP-<nowiki>#</nowiki>20_SP
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**#36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
+
**<nowiki>#</nowiki>36-3-29-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1AK3_EP

Revision as of 10:29, 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 (gene, 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~

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

    • #36-3-14-5-2-3-17-5 from 0911 MP product
  • 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)

Retrieved from "http://2011.igem.org/Team:UT-Tokyo/LabNote"