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 == | ||
- | [[File: | + | [[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 == | ||
- | [[File: | + | [[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 == | ||
- | [[File: | + | [[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 == | ||
- | [[File: | + | [[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 == | ||
- | [[File: | + | [[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
- 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
- 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
- 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
- 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
- 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