Team:Heidelberg/Templates/MM week10

From 2013.igem.org

(Difference between revisions)
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== 2013-07-01 ==
== 2013-07-01 ==
* finish [[Isopropanol PCR purification|purification]] (digest for 5h, dissolve in 50 µl H<sub>2</sub>O) -> 1162 ng/µl (performed by Fanny)
* finish [[Isopropanol PCR purification|purification]] (digest for 5h, dissolve in 50 µl H<sub>2</sub>O) -> 1162 ng/µl (performed by Fanny)
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== 2013-07-02 ==
== 2013-07-02 ==
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[[File:Heidelberg_BAP1-colony-PCR2013-07-02.png|300px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2, 3: negative control (BAP1-pLF03); lanes 4-9: colony-PCR of F-rest (lanes 8,9: from lawn); lanes 10-13: colony-PCR of H-10µl; lanes 2,4,6,8,10,12: primers IK01+IK02; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control) ]]
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[[File:Heidelberg_Heidelberg_BAP1-colony-PCR2013-07-02.png|300px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2, 3: negative control (BAP1-pLF03); lanes 4-9: colony-PCR of F-rest (lanes 8,9: from lawn); lanes 10-13: colony-PCR of H-10µl; lanes 2,4,6,8,10,12: primers IK01+IK02; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control) ]]
* bacteria lawn on rest-plate from -80°C, possible colonies on other plates (barely visible, may be bubbles)
* bacteria lawn on rest-plate from -80°C, possible colonies on other plates (barely visible, may be bubbles)
* pick 2 colonies from F-rest, sample of lawn, 2 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control) (20µl total volume):
* pick 2 colonies from F-rest, sample of lawn, 2 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control) (20µl total volume):
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== 2013-07-03 ==
== 2013-07-03 ==
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[[File:Heidelberg_BAP1-colony-PCR2013-07-03.png|300px|thumb|left|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.<br/>
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[[File:Heidelberg_Heidelberg_BAP1-colony-PCR2013-07-03.png|300px|thumb|left|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.<br/>
Lane 1: NEB 2-log<br/>
Lane 1: NEB 2-log<br/>
Top: lanes 2-4: negative control (BAP1-pLF03); lanes 5-13: colony-PCR F-10µl<br/>
Top: lanes 2-4: negative control (BAP1-pLF03); lanes 5-13: colony-PCR F-10µl<br/>
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== 2013-07-04 ==
== 2013-07-04 ==
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[[File:Heidelberg_BAP1-colony-PCR2013-07-04.png|300px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.<br/>
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[[File:Heidelberg_Heidelberg_BAP1-colony-PCR2013-07-04.png|300px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.<br/>
Lane 1: NEB 2-log<br/>
Lane 1: NEB 2-log<br/>
Top: lanes 2-10: cultures from 2013-07-03; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03; lanes 4,7,10: primers IK05+IK06; lane 11: BAP1-pLF03 (negative control); lanes 12-13: colonies from F-10µl plate; lanes 11-13: primers IK05+IK06<br/>
Top: lanes 2-10: cultures from 2013-07-03; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03; lanes 4,7,10: primers IK05+IK06; lane 11: BAP1-pLF03 (negative control); lanes 12-13: colonies from F-10µl plate; lanes 11-13: primers IK05+IK06<br/>
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== 2013-07-05 ==
== 2013-07-05 ==
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[[File:Heidelberg_PKD46 digest 2013-07-05.png|100px|thumb|left|Restriction digest of pKD46. Lane 1: NEB 2-log; lane 2: EcoRI; lane 3: BamHI+NotI; lane 4: PstI+NotI; lane 5: undigested (225 ng (5 ml) loaded)]]
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[[File:Heidelberg_Heidelberg_PKD46 digest 2013-07-05.png|100px|thumb|left|Restriction digest of pKD46. Lane 1: NEB 2-log; lane 2: EcoRI; lane 3: BamHI+NotI; lane 4: PstI+NotI; lane 5: undigested (225 ng (5 ml) loaded)]]
* digest 450 ng pKD46 (10 µl of 45.5 and 46 ng/µl miniPreps from 2013-06-06) with EcoRI (expected: 4816+1505 bp), BamHI+NotI (expected: 3675+2646 bp), PstI+NotI (expected: 3711+2363+243 bp); 30 µl total volume
* digest 450 ng pKD46 (10 µl of 45.5 and 46 ng/µl miniPreps from 2013-06-06) with EcoRI (expected: 4816+1505 bp), BamHI+NotI (expected: 3675+2646 bp), PstI+NotI (expected: 3711+2363+243 bp); 30 µl total volume
* right plasmid
* right plasmid
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== 2013-07-06 ==
== 2013-07-06 ==
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[[File:Heidelberg_BAP1-pKD46+pLF03-PCR digest 2013-07-06.png|100px|thumb|right|Restriction digest of BAP1-pKD46 electroporated with pLF03-PCR grown in Cm+IPTG(1mM), miniprepped.<br/>
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[[File:Heidelberg_Heidelberg_BAP1-pKD46+pLF03-PCR digest 2013-07-06.png|100px|thumb|right|Restriction digest of BAP1-pKD46 electroporated with pLF03-PCR grown in Cm+IPTG(1mM), miniprepped.<br/>
Lane 1: NEB 2-log; lanes 2-5: Konrad's PCR; lane 6: digested with EcoRI; lane 8: undigested]]
Lane 1: NEB 2-log; lanes 2-5: Konrad's PCR; lane 6: digested with EcoRI; lane 8: undigested]]
* add Cm (1:3000), IPTG (1:1000) to liquid cultures, grow for 8h at 30°C
* add Cm (1:3000), IPTG (1:1000) to liquid cultures, grow for 8h at 30°C

Revision as of 22:46, 4 October 2013

Contents

2013-07-01

  • finish purification (digest for 5h, dissolve in 50 µl H2O) -> 1162 ng/µl (performed by Fanny)
  • prepare electrocompetent cells from ON culture (add 0.5% arabinose at dilution) (performed by Fanny)
  • electroporate fresh electrocompetent cells, 1 aliquot from 2013-06-26 (23 µl DNA each) (performed by Fanny)
  • grow in SOC at 30°C for 30 min, transfer to falcons, add 1 ml LB, add IPTG (1 mM), grow at 37°C for 4h
  • spin cell down (8000 rcf for 3 min), decant supernatant, resuspend pellet in remaining medium
  • plate on Cm+IPTG (2 plates for each sample: 10µl + rest, plates marked F: from -80°C, marked H: newly prepared cells), grow at 37°C

2013-07-02

File:Heidelberg Heidelberg BAP1-colony-PCR2013-07-02.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2, 3: negative control (BAP1-pLF03); lanes 4-9: colony-PCR of F-rest (lanes 8,9: from lawn); lanes 10-13: colony-PCR of H-10µl; lanes 2,4,6,8,10,12: primers IK01+IK02; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control)
  • bacteria lawn on rest-plate from -80°C, possible colonies on other plates (barely visible, may be bubbles)
  • pick 2 colonies from F-rest, sample of lawn, 2 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control) (20µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • very evening: colonies on F-10µl, H-rest, H-10µl plates appear -> leave at 37°C ON

2013-07-03

File:Heidelberg Heidelberg BAP1-colony-PCR2013-07-03.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-4: negative control (BAP1-pLF03); lanes 5-13: colony-PCR F-10µl
bottom:lanes 2-4: colony-PCR of F-10µl; lanes 5-13: colony-PCR of H-10µl; lanes 2,5,11: primers IK01+IK02; lanes 3,6,12: primers IK01+IK03 (positive control); lanes 4,7,13: primers IK05+IK06 (negative control)
  • colonies on F-rest plate appeared (plate left ON at RT)
  • pick 4 colonies from F-10µl, 3 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control), IK05+IK06 (negative control):
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • F-10µl 1,2,4 show no negative control -> grow at 37°C
  • continue growing plates at 37°C until evening, leave at RT ON

2013-07-04

File:Heidelberg Heidelberg BAP1-colony-PCR2013-07-04.png
Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-10: cultures from 2013-07-03; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03; lanes 4,7,10: primers IK05+IK06; lane 11: BAP1-pLF03 (negative control); lanes 12-13: colonies from F-10µl plate; lanes 11-13: primers IK05+IK06
bottom: lanes 2-13: primers IK05+IK06; lanes 2,3: colonies from F-10µl plate; lanes 4-6: colonies from F-rest plate; lanes 7-10: colonies from H-10µl plate; lanes 11-13: colonies from H-rest plate
  • repeat colony-PCR for 3 ambiguous cultures form 2013-07-03 (use 1 µl of culture), pick 14 new colonies (only primers IK05+IK06); 20 µl total volume:
Cycles temperature [°C] Time [s]
1 95 300
12 95 60
68 ↓0.5°C 30
72 120
18 95 60
62 30
72 120
1 72 600
1 4 inf
  • no positives

2013-07-05

File:Heidelberg Heidelberg PKD46 digest 2013-07-05.png
Restriction digest of pKD46. Lane 1: NEB 2-log; lane 2: EcoRI; lane 3: BamHI+NotI; lane 4: PstI+NotI; lane 5: undigested (225 ng (5 ml) loaded)
  • digest 450 ng pKD46 (10 µl of 45.5 and 46 ng/µl miniPreps from 2013-06-06) with EcoRI (expected: 4816+1505 bp), BamHI+NotI (expected: 3675+2646 bp), PstI+NotI (expected: 3711+2363+243 bp); 30 µl total volume
  • right plasmid
  • inoculate 2 x 7 ml LB+Cm+IPTG(1mM) with colonies from F-10µl plate, grow at 30°C

2013-07-06

File:Heidelberg Heidelberg BAP1-pKD46+pLF03-PCR digest 2013-07-06.png
Restriction digest of BAP1-pKD46 electroporated with pLF03-PCR grown in Cm+IPTG(1mM), miniprepped.
Lane 1: NEB 2-log; lanes 2-5: Konrad's PCR; lane 6: digested with EcoRI; lane 8: undigested
  • add Cm (1:3000), IPTG (1:1000) to liquid cultures, grow for 8h at 30°C
  • make miniPreps -> 25 ng/µl and 14.4 ng/µl in 27.5 µl
  • digest 25 ng/µl miniPrep with EcoRI (use all of miniPrep)
  • load digest completely, 10 µl of 14.4 ng/µl miniPrep on gel
  • no bands -> nanoDrop crappy, no plasmid
  • wrong strain? Streak BAP1 on Cm, grow at 37°C