Team:Heidelberg/Templates/MM week11
From 2013.igem.org
(Difference between revisions)
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== 2013-07-08 == | == 2013-07-08 == | ||
- | [[File:Heidelberg_PLF03_PCR2013-07-08.png|100px|thumb | + | [[File:Heidelberg_PLF03_PCR2013-07-08.png|100px|thumb|gel electrophoresis of PCR of pLF03 with primers IK07 and IK08 using Q5 polymerase. Product from one PCR tube was split to two neighboring lanes.]] |
[[File:Heidelberg_PLF03_PCR2013-07-08_gel-extracted.png|100px|thumb|right|1 µl of gel-extracted PCR amplificate was loaded. Lane 1: NEB 2-log; lane 2: amplificate extracted by Dominik, lane 3: amplificate extracted by Ilia]] | [[File:Heidelberg_PLF03_PCR2013-07-08_gel-extracted.png|100px|thumb|right|1 µl of gel-extracted PCR amplificate was loaded. Lane 1: NEB 2-log; lane 2: amplificate extracted by Dominik, lane 3: amplificate extracted by Ilia]] | ||
* no colonies of BAP1 on Cm | * no colonies of BAP1 on Cm | ||
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== 2013-07-10 == | == 2013-07-10 == | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-10.png|300px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-10.png|300px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lanes 2, 13: BAP1-pLF03 (control); lanes 3-12: colony-PCRs of large colonies from 25µl/10µl plate. Lanes 2-12: OneTaq; lane 13: iTaq]] |
* on all plates: large + small colonies | * on all plates: large + small colonies | ||
* small colonies too small to pick => pick 10 large colonies from 25µl DNA / 10 µl bacteria plate, run colony-PCR with primers IK05+IK06 (Taq, 20 µl total volume): | * small colonies too small to pick => pick 10 large colonies from 25µl DNA / 10 µl bacteria plate, run colony-PCR with primers IK05+IK06 (Taq, 20 µl total volume): | ||
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== 2013-07-11 == | == 2013-07-11 == | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-11.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-11.png|100px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB at 37°C. Lanes 1,4: primers IK01+IK03 (positive control); lanes 2,5: primers IK01+IK02; lanes 3,6: primers IK05+IK06; lanes 1-3: BAP1-pLF03 (control); lanes 4-6: colony 7; lane 7: NEB 2-log]] |
* run full colony-PCR of liquid culture (OneTaq, 20 µl total volume, use 1 µl of culture): | * run full colony-PCR of liquid culture (OneTaq, 20 µl total volume, use 1 µl of culture): | ||
{| class="wikitable" | {| class="wikitable" | ||
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* no integration at all | * no integration at all | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-11_3.png|300px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-11_3.png|300px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06.<br/> |
Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-9: colonies from 1µl/10µl plate; lanes 10-13: colonies from 1µl/rest plate<br/> | Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-9: colonies from 1µl/10µl plate; lanes 10-13: colonies from 1µl/rest plate<br/> | ||
Bottom: lane 1: NEB 2-log; lanes 2-10: colonies from 1µl/rest plate (lane 5: swiped 3rd population of extremely small colonies)]] | Bottom: lane 1: NEB 2-log; lanes 2-10: colonies from 1µl/rest plate (lane 5: swiped 3rd population of extremely small colonies)]] | ||
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* no bands | * no bands | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-12_2.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-12_2.png|100px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lanes 3-4: colony-PCRs (from colonies marked 1 and 2)]] |
* run colony-PCR with primers IK05+IK06 of 2 colonies from liquid culture that were transferred to Cm + IPTG plate (iTaq, 20µl total volume): | * run colony-PCR with primers IK05+IK06 of 2 colonies from liquid culture that were transferred to Cm + IPTG plate (iTaq, 20µl total volume): | ||
{| class="wikitable" | {| class="wikitable" | ||
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== 2013-07-14 == | == 2013-07-14 == | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-14_1.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-14_1.png|100px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR]] |
* bacteria grew on Amp => 3 possibilities: | * bacteria grew on Amp => 3 possibilities: | ||
** Amp plates not selective | ** Amp plates not selective |
Latest revision as of 02:22, 5 October 2013
Contents |
2013-07-08
- no colonies of BAP1 on Cm
- repeat PCR of pLF03:
- run 4 PCRs of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (50 µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
45 | 98 | 5 |
66 | 30 | |
72 | 150 | |
1 | 72 | 1800 (30 min) |
1 | 4 | inf |
- extract amplificate from gel (4 lanes pooled, extracted by Dominik -> 119 ng/µl; 4 lanes pooled, extracted by Ilia -> 111.5 ng/µl)
- load 1 µl of each extract on gel
2013-07-09
- electroporate electrocompetent BAP1-pKD46 with 1µl, 5µl, 25µl DNA
- resuspend in 1 ml SOC + IPTG (1mM) + Ara (0.1%)
- grow for 90 min at 37°C, 400 rpm
- spin cells down, plate 10µl, rest of each sample on Cm+IPTG
2013-07-10
- on all plates: large + small colonies
- small colonies too small to pick => pick 10 large colonies from 25µl DNA / 10 µl bacteria plate, run colony-PCR with primers IK05+IK06 (Taq, 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 integration
- continue growing plates at 37°C
- when small colonies large enough: pick, run colony-PCR (OneTaq, 20µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 120 | |
23 | 95 | 60 |
62 | 30 | |
72 | 120 | |
1 | 72 | 600 |
1 | 4 | inf |
- 1st colony from 5µl/rest plate (marked 7) seems promising => grow at 37°C in 1 ml LB
2013-07-11
- run full colony-PCR of liquid culture (OneTaq, 20 µl total volume, use 1 µl of culture):
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 integration
- 3rd population of colonies appeared on plates (stored at RT): very small, probably satellites
- run colony-PCR of liquid with primers IK07+IK08 (test for integration in wrong place) (OneTaq, 20µl total volume, use 1 µl liquid culture):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
30 | 95 | 60 |
66 | 30 | |
72 | 600 | |
1 | 72 | 600 |
1 | 12 | inf |
- no integration at all
- pick 20 new colonies, run colony-PCR with primers IK05+IK06 (iTaq, 20 µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 120 | |
23 | 95 | 60 |
62 | 30 | |
72 | 120 | |
1 | 72 | 600 |
1 | 4 | inf |
- unspecific bands (also present in colony-PCR from 2013-07-10)
- unspecific bands not present in BAP1-pLF03 PCR (control) and bottom lane 5 (satellite colonies) => not due to iTaq
- assume: some amount of satellites was picked together with colonies from plates -> give band at 500 bp; partially amplified ygfG fragments prime integrated insert
- do multiple sequence alignment of ygfG, [http://www.ncbi.nlm.nih.gov/nuccore/AF113605 pccB], [http://www.ncbi.nlm.nih.gov/nuccore/AF113603 accA1], and [http://www.ncbi.nlm.nih.gov/nuccore/AY048742 catR] using ClustalO => long enough stretches of partially identical sequences present
- add 1mM IPTG to cultures with colonies from 1µl/10µl plate picked today (grew in 500 µl LB at 37°C for several hours), after 2h at 37°C: streak on Cm+IPTG
- pick 10 colonies each from 1µl/10µl and 1µl/rest plates, transfer to new Cm+IPTG plate, grow at 37°C
- pick 1 colony from 1µl/10µl plate, inoculate 1 ml LB + Cm + IPTG (1mM)
2013-07-12
- colonies grew slowly
- run colony-PCR with primers IK01+IK02 (iTaq, 20 µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 120 | |
23 | 95 | 60 |
62 | 30 | |
72 | 120 | |
1 | 72 | 600 |
1 | 4 | inf |
- no bands
- run colony-PCR with primers IK05+IK06 of 2 colonies from liquid culture that were transferred to Cm + IPTG plate (iTaq, 20µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 120 | |
23 | 95 | 60 |
62 | 30 | |
72 | 120 | |
1 | 72 | 600 |
1 | 4 | inf |
- band at 1.2 kb is now specific -> WTF?!?
2013-07-13
- need to determine whether 1.2 kb band is specific: run gradient PCR of colony 2 (iTaq, 20 µl total volume) with primers IK05+IK06:
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
35 | 95 | 60 |
63-68 (ΔT = 1) | 30 | |
72 | 120 | |
1 | 72 | 600 |
1 | 4 | inf |
- intensity of 1.2kb band decreases, intensity of 0.5 kb band (control) does not -> 1.2 kb band is unspecific product
- check for presence of genomically integrated insert: run colony-PCR of colony 2 with primers IK07+IK08 (iTaq, 20 µl total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
35 | 95 | 60 |
66 | 30 | |
72 | 600 | |
1 | 72 | 600 |
1 | 12 | inf |
- transfer some of colony 2 on Amp (check for presence of pLF03 as a whole), grow at 37°C
- transfer some of colony 2 to liquid culture: LB + Cm + IPTG (1mM), grow at 37°C
2013-07-14
- bacteria grew on Amp => 3 possibilities:
- Amp plates not selective
- bacteria have complete pLF03 plasmid
- bacteria still have pKD46, although grown at 37°C
- plate BAP1, BAP1-pKD46 on Amp, grow at 37°C
- colony-PCR did not work for the control -> repeat with OneTaq (use 1 µl of liquid culture)
- no amplificate for colony: possibly, Taq has problems amplifying 4.8 kb from genomic DNA
- run colony-PCR with primers IK01+IK06 (OneTaq, 20 µl total volume, use 1 µl of liquid culture):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 240 | |
23 | 95 | 60 |
62 | 30 | |
72 | 240 | |
1 | 72 | 600 |
1 | 4 | inf |
- control shows bright band where expected, colony does not => something is integrated, unknown what