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Team:Kyoto/Notebook
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==Notebook== | ==Notebook== | ||
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===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>=== | ===Tuesday, July 20 <span class="by">By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto</span>=== | ||
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Revision as of 17:13, 5 October 2010
Index
Notebook
Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
Solubilization of Antibiotics (Ampicillin, Kanamycin)
| Ampicillin(Amp) | Kanamycin(Kan) | |
| Mix 1.0g Amp and 20ml MilliQ | Mix 0.5g Kan and 10ml MilliQ | Final concentration is 50mg/ml |
| Dispense 1.1ml of the solution into 1.5ml tubes | ||
| Store in the freezer (-20℃) | ||
Making plates for LB (Amp+) and LB (Kan+)
Transformation
| Name | Well | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|---|
| <partinfo>J23100</partinfo> | 1-18-C | 1 | 20 | 21 | LB (Amp+) | At 37℃, 7/20 20:50 - 7/21 17:00 | ○ |
| <partinfo>J23105</partinfo> | 1-18-M | 1 | 20 | 21 | ○ | ||
| <partinfo>J23116</partinfo> | 1-20-M | 1 | 20 | 21 | ○ | ||
| <partinfo>R0011</partinfo> | 1-6-G | 1 | 20 | 21 | ○ | ||
| <partinfo>E0840</partinfo> | 1-12-O | 1 | 20 | 21 | ○ | ||
| <partinfo>J06702</partinfo> | 2-8-E | 1 | 20 | 21 | ○ | ||
| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | × | ||
| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | LB (Kan+) | × |
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance. So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
Wednesday, July 21 By: Wataru, Ken, Makoto, Takuya Y.
Culture of plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00
Making a master plate of the above plates
Retry Transformation
| Name | Well | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|---|
| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | LB (Kan+) | At 37℃ 7/21 20:50 - 7/22 16:30 | ○ |
| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | ○ |
PCR for S-R-Rz/Rz1 and S
| No. | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | TemplateDNA (5ng/µl) | Primer Forward (10µM) | Primer S-R-Rz/Rz1 Reverse (10µM) | Primer S Reverse (10µM) | KOD Plus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl |
| 2 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl |
| 3 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl |
| 4 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl |
| 5 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl |
| 6 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl |
| 7 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl |
| 8 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl |
| 94℃ | 2min | |
| 98℃ | 10sec | 30 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
Thursday, July 22 By: Wataru
Electrophoresis of the PCR products for 40min
Length of S and S-R-Rz/Rz1 is 370bp and 1300bp, so PCR succeeded.
Miniprep
| Name | Concentration(ng/µl) |
|---|---|
| <partinfo>J23100</partinfo> | 18.5 |
| <partinfo>J23105</partinfo> | 12.5 |
| <partinfo>J23116</partinfo> | 14.6 |
| <partinfo>R0011</partinfo> | 8.6 |
| <partinfo>E0840</partinfo> | 12.1 |
| <partinfo>J06702</partinfo> | 14.7 |
The concentration of all samples was very week. Probably our shaking incubation was week.
Culture of plates and making master plates of <partinfo>pSB4K5</partinfo> and <partinfo>B0015</partinfo> from 07/22 17:00 to 07/23 10:00
Friday, July 23 By: Wataru, Tomo, Makoto
Miniprep
| Name | Concentration(ng/µl) |
|---|---|
| <partinfo>pSB4K5</partinfo> | 79.2 |
| <partinfo>B0015</partinfo> | - |
We lost <partinfo>B0015</partinfo> by our mistake. The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
Picking up number 1, 3, 5, and 7 of the products of PCR, and PCR-purification
| No. | Name | Concentration (ng/µl) | New Name |
|---|---|---|---|
| 1 | S-R-Rz/Rz1 | 18.6 | - |
| 3 | S | 77.6 | S1 |
| 5 | S-R-Rz/Rz1 | 33.6 | - |
| 7 | S | 65.4 | S2 |
The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
Retry of Standard PCR for S-R-Rz/Rz1
| No. | Water | 25mmol/l MgSO4 | 2mmol/l dNTPs | 10×Buffer for KOD plus ver.2 | Template DNA (5ng/µl) | Primer S-R-Rz/Rz1 Forward (10µmol/l) | Primer S-R-Rz/Rz1 Reverse (10µmol/l) | KOD plus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 28µl | 3 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 2 | 28 | 3 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 3 | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 4 | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 5 | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 6 | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 30 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
Restriction Digestion of <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, and PstI to check function of our Restriction Enzyme
| No. | 10xBuffer | BSA | Enzyme | MilliQ | Total | Incubation |
|---|---|---|---|---|---|---|
| 1 | 5µl | 1 | EcoRI 0.1 | 3.6 | 10 | At 37℃ 7/23 18:00 - 7/23 18:30 |
| 2 | 5 | 1 | XbaI 0.1 | 3.6 | 10 | |
| 3 | 5 | 1 | SpeI 0.1 | 3.6 | 10 | |
| 4 | 5 | 1 | PstI 0.1 | 3.6 | 10 | |
| 5 | 5 | 1 | - | 3.7 | 10 |
Electrophoresis of above sample for 35min
Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.
Digestion of the PCR products of S gene by EcoRI and SpeI and <partinfo>E0840</partinfo> by EcoRI and XbaI to insert S gene to <partinfo>E0840</partinfo>
| Name | Sample Volume (µl) | 10×Buffer | Enzyme 1 | Enzyme 2 | MilliQ | Total | Incubation |
|---|---|---|---|---|---|---|---|
| S1 | 11 | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50 | At 37℃ for 2h |
| S2 | 11 | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50 | |
| <partinfo>E0840</partinfo> | 45 | 5 | EcoRI 0.2 | XbaI 0.2 | 0 | 50 |
After PCR purification, evaporated them and diluted 3ul.
Ligation
| Name | Vector | Insert | Ligation High | Total |
|---|---|---|---|---|
| S-E08401 | <partinfo>E0840</partinfo> 0.5µl | S1 0.5 | 1 | 2 |
| S-E08402 | <partinfo>E0840</partinfo> 0.5 | S2 0.5 | 1 | 2 |
Monday, July 26 By: Wataru, Tomonori, Makoto
Electrophoresis of PCR Products
At the condition 4 (4.5µl MgSO4) and 6 (6µl MgSO4), S-R-Rz/Rz1 is amplified very much. So we decided to use them.
PCR Purification
| No. | Concentration (ng/µl) | New Name |
|---|---|---|
| 4 | 51.6 | SRRz1 |
| 5 | 59.3 | |
| 6 | 59.6 | SRRz2 |
Transformation
| Name | Well | Sample (µl) | Competent Cell (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|---|
| <partinfo>E0240</partinfo> | 1-12-M | 1 | 20 | 21 | LB (Amplicillin+) | At 37℃ 7/26 - 7/27 | × |
| <partinfo>I20260</partinfo> | 2-17-F | 1 | 20 | 21 | LB (Kanamycin+) | × | |
| <partinfo>J04450</partinfo> | 1-5-E | 1 | 20 | 21 | × |
Culture of <partinfo>pSB4K5</partinfo>, <partinfo>E0840</partinfo>, and <partinfo>B0015</partinfo>
Tuesday, July 27 By: Wataru, Tomo, Kazuya, Ken, Naoi
Team:Kyoto/Protocols#Colony_PCR~Colony PCR of S-E0840 (Electrophoresis for 35min)
| Marker | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | + | - | Marker |
| 1kb | S-E08401 | S-E08402 | E0840 | None | 100bp | |||||||||||
As a result, 1, 3, 5, 6, 11, 12, and 13 are inserted S gene correctly. So, we decided to use 6 as S-E08401 and 11 as S-E08402.
Miniprep
| Name | Concentration(ng/µL) |
|---|---|
| <partinfo>R0011</partinfo> | 26.9 |
| <partinfo>B0015</partinfo> | 120.0 |
| <partinfo>E0840</partinfo> | 120.1 |
Restriction Digestion
| Sample volume | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | Incubation | |||
|---|---|---|---|---|---|---|---|---|---|---|
| <partinfo>B0015</partinfo> | 30 | 5 | 0.5 | EcoRI | 0.4 | XbaI | 0.3 | 13.7 | 50 | At 37℃ 16:45 - 18:00 |
| SRRz1 | 40 | 5 | 0.5 | 0.4 | 0.4 | 3.8 | 50 | |||
| SRRz2 | 40 | 5 | 0.5 | 0.4 | 0.4 | 3.8 | 50 | |||
Transformation
| Name | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|
| SRRz1-B0015 | rowspan="2" | rowspan="2" | ○ | |||
| SRRz2-B0015 | ○ |
Wednesday, July 28 By:
Miniprep
| Name | Concentration(ng/µl) |
|---|---|
| S-E08401 | 95.5 |
| S-E08402 | 98.6 |
Diluted S-E08401 and S-E08402 20 times with water, and used as template DNA.
Deletion_PCR to delete a functional domain of S gene
| Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | Primer Forward(10µM) | Primer Reverse(10µM) | Template S-E08401 | Template S-E08402 | KOD Plus ver.2 | Total | |
|---|---|---|---|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 28 | 3 | 5 | 5 | 1.5 | 1.5 | 5 | - | 1 | 50 |
| SΔTMD1-E08401-2 | 28 | 3 | 5 | 5 | 1.5 | 1.5 | 5 | - | 1 | 50 |
| SΔTMD1-E08402 | 28 | 3 | 5 | 5 | 1.5 | 1.5 | - | 5 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 35 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
Restriction Digestion to check the function of DpnI
| Name | Sample | fast digestion buffer | DpnI | MilliQ | Total |
|---|---|---|---|---|---|
| S-E08401 | 3 | 1 | 0.1 | 5.8 | 10 |
| S-E08402 | 3 | 1 | 0.1 | 5.8 | 10 |
Electrophoresis for 35min
| Marker | 1 | 2 | 3 | 4 | Marker |
| 1kb | Not digested S-E08401 | Not digested S-E08402 | Digested S-E08401 | Digested S-E08402 | 100bp |
DpnI works correctly.
Thursday, July 29 By:
Restriction Digestion
| Name | Sample volume | Fastdigestion Buffer | Enzyme 1 | MilliQ | Total | Incubation | |
|---|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 50 | 6 | DpnI | 0.2 | 3.8 | 60 | 07/29 09:40 - 07/29 11:00 |
| SΔTMD1-E08402 | 50 | 6 | DpnI | 0.2 | 3.8 | 60 | |
Ligation and Pospholylation
| Name | Sample | MilliQ | Ligation High | T4 Kinase | Total | Incubation |
|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 2 | 7 | 5 | 1 | 15 | 07/29 11:30 ~ 07/29 13:00 |
| SΔTMD1-E08402 | 2 | 7 | 5 | 1 | 15 |
Transformation
| Name | Sample Volume(µL) | Competent Cell(µL) | Total | Plate | Incubation | Result |
|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 3 | 30 | 33 | LB Amp+ | 07/29 ~ 07/30 | ○ |
| SΔTMD1-E08402 | 3 | 30 | 33 | ○ |
Monday, August 2 By: Wataru, Ken
Miniprep
| Name | Concentration(ng/µL) |
|---|---|
| SΔTMD1-E0840-1 | 52.7 |
| SΔTMD1-E0840-2 | 54.4 |
| SΔTMD1-E0840-3 | 89.5 |
| <partinfo>pSB4K5</partinfo> | 50.7 |
| <partinfo>R0011</partinfo> | 18.6 |
Standard PCR of <partinfo>E0240</partinfo>
E240 is very important parts to measure RPU of promoters in iGEM. However, we failed to transfect it to E.coli from parts kit of iGEM. So we decided to amplify this parts by PCR.
| Name | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | Primer VF2(10µM) | Primer VR(10µM) | Template E240 | KOD Pllus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|
| E02401 | 28 | 3 | 5 | 5 | 1.5 | 1.5 | 5 | 1 | 50 |
| E02402 | 28 | 3 | 5 | 5 | 1.5 | 1.5 | 5 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 35 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
PCR Purification
| Sample number | Concentration(ng/µL) |
|---|---|
| E02401 | 42.6 |
| E02402 | 55.3 |
Restriction Digestion for inserting <partinfo>E0240</partinfo> to pSB4K5 by 3A assembly
| Name | Sample volume | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | ||
|---|---|---|---|---|---|---|---|---|---|
| E02401(X-P) | 30 | 5 | 0.5 | XbaI | 0.2 | PstI | 0.2 | 14.1 | 50 |
| E02402(X-P) | 30 | 5 | 0.5 | XbaI | 0.2 | PstI | 0.2 | 14.1 | 50 |
PCR Purification
| Name | Concentration(ng/µL) | Volume(µL) |
|---|---|---|
| E02401(X-P) | 21.8 | 40 |
| E02402(X-P) | 32.4 | 45 |
Stored at -20℃.
Error PCR
| Name | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | Primer VF2(10µM) | Primer VR(10µM) | Template Δ1 | Template | Template | KOD Pllus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | - | - | 1 | 50 |
| SΔTMD1-E08401-2 | 32 | 3 | 5 | 5 | 1.5 | 1.5 | - | 1 | - | 1 | 50 |
| SΔTMD1-E08402 | 32 | 3 | 5 | 5 | 1.5 | 1.5 | - | - | 1 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 20 cycles |
| 68℃ | 4min | |
| 4℃ | forever |
Transformation
| Name | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|
| SΔTMD1-E08401-1 | 2 | 20 | 22 | rowspan="3" | rowspan="3" | ○ |
| SΔTMD1-E08401-2 | 2 | 20 | 22 | } | ||
| SΔTMD1-E08402 | 2 | 20 | 22 | ○ |
Tuesday, August 3 By:
=Culture of each two colonies of SΔTMD1-E08401-1 and SΔTMD1-E08402 for 37℃ 08/03-08/04
Miniprep for Construction of Measure(lacP) and Measure(Standard)
| Sample number | Concentration(ng/µL) |
|---|---|
| <partinfo>pSB4K5</partinfo> | 60.7 |
| <partinfo>R0011</partinfo> | 26.8 |
Restriction Digestion
| Name | Sample volume | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | ||
|---|---|---|---|---|---|---|---|---|---|
| R0011 | 50 | 6 | 0.6 | EcoRI | 0.2 | SpeI | 0.2 | 3 | 60 |
| pSB4K5(E-P) | 50 | 6 | 0.6 | EcoRI | 0.2 | PstI | 0.2 | 3 | 60 |
| E02401(X-P) | 50 | 6 | 0.6 | XbaI | 0.2 | PstI | 0.2 | 3 | 60 |
| E02402(X-P) | 50 | 6 | 0.6 | XbaI | 0.2 | PstI | 0.2 | 3 | 60 |
PCR Purification
| Sample number | Concentration(ng/µL) |
|---|---|
| pSB4K5(E-P) | 39.5 |
| E02401(X-P) | 21.8 |
| E02402(X-P) | 32.4 |
pSB4K5(E-P) is concentrated 10µL and E02401(X-P), E02402(X-P) are concentrated 1µL.
Ethanol Precipitation
Dilution of <partinfo>pSB4K5</partinfo> by 2µl MilliQ
Ligation
| Vector | Insert 1 | Insert 2 | Ligation High | Total | Incubation | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| R0011-E02401[Low] | pSB4K5(E-P) | 1 | R0011(E-S) | 1 | E02401(X-P) | 1 | 3 | 15 | 17:30 - 20:20 | |
| R0011-E02402[Low] | pSB4K5(E-P) | 1 | R0011(E-S) | 1 | E02402(X-P) | 1 | 3 | 15 | ||
Standard PCR of <partinfo>J23101</partinfo>-<partinfo>E0240</partinfo> that is important in the measurement of RPU
| Name | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | Primer VF2(10µM) | Primer VR(10µM) | Template J23101-E0240 | KOD plus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|
| J23101-E02401 | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
| J23101-E02402 | 32 | 3 | 5 | 5 | 1.5 | 1.5 | - | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 30 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
PCR Purification
| Name | Concentration(ng/µL) |
|---|---|
| J23101-E0240 | 40.6 |
Restriction Digestion
| Name | Sample volume | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | ||
|---|---|---|---|---|---|---|---|---|---|
| J23101-E0240(E-P) | 45 | 6 | 0.6 | EcoRI | 0.2 | PstI | 0.2 | 8 | 60 |
PCR Purification
| Name | Concentration(ng/µL) | Volume(µL) |
|---|---|---|
| J23101-E0240(E-P) | 74.1 | 30 |
J23101-E0240(E-P) is concentrated 7µL
Ligation
| Vector | Insert | Ligation High | Total | Incubation | |||
|---|---|---|---|---|---|---|---|
| J23101-E0240[Low] | pSB4K5(E-P) | 1 | J23101-E0240(E-P) | 1 | 2 | 4 | 20:00-20:30 |
Transformation
| Name | Conc(/µL) | Sample Volume(µL) | Competent Cell(µL) | Total | Plate | Incubation |
|---|---|---|---|---|---|---|
| R0011-E02401[Low] | - | 1 | 20 | 21 | LB kan | 8/3~8/4 |
| R0011-E02402[Low] | - | 1 | 20 | 21 | ||
| J23101-E0240[Low] | - | 1 | 20 | 21 |
Thursday, August 5 By:
Result of Transformation
| R0011-E02401[Low] | Many colonies |
| R0011-E02402[Low] | |
| J23101-E0240[Low] |
pSB4K5 is inserted RFP generator. We didn't distinguish this inserted parts from low copy plasmid backbone, so self-ligated colony is red. So, white colony is correctly inserted parts.
However, white colonies and green colonies are observed in R0011-E02401[Low] and R0011-E02402[Low] plate. We cultured both white and green colonies.
In J23101-E0240[Low], Many of colonies are red, but green colonies are observed. We cultured green colonies.
| Name | Color | Incubation |
|---|---|---|
| R0011-E02401[Low]-1 | Green Colony | 8/5-8/6 |
| R0011-E02401[Low]-2 | Green Colony | |
| R0011-E02401[Low]-3 | White Colony | |
| R0011-E02401[Low]-4 | White Colony | |
| R0011-E02402[Low]-1 | Green Colony | |
| R0011-E02402[Low]-2 | White Colony | |
| R0011-E02402[Low]-3 | White Colony | |
| R0011-E02402[Low]-4 | White Colony | |
| J23101-E0240[Low]-1 | Green Colony | |
| J23101-E0240[Low]-2 | Green Colony | |
| J23101-E0240[Low]-3 | Green Colony |
| Name | Concentration(ng/µL) |
|---|---|
| SΔTMD1-E08401-1-A | 28.9 |
| SΔTMD1-E08401-1-B | 25.3 |
| SΔTMD1-E08402-A | 26.6 |
| SΔTMD1-E08402-B | 24.0 |
As a result, deletion is succeeded, however, point mutation is failed. It is because DpnI is too little to digest all of template DNA.
Friday, August 6
Miniprep
| Name |
|---|
| R0011-E02401[Low]-1 |
| R0011-E02401[Low]-2 |
| R0011-E02401[Low]-3 |
| R0011-E02401[Low]-4 |
| R0011-E02402[Low]-1 |
| R0011-E02402[Low]-2 |
| R0011-E02402[Low]-3 |
| R0011-E02402[Low]-4 |
| J23101-E0240[Low]-1 |
| J23101-E0240[Low]-2 |
| J23101-E0240[Low]-3 |
Restriction Digestion
| Name | Sample volume | 2 buffer | BSA | Enzyme 1 | Enzyme 2 | MilliQ | Total | ||
|---|---|---|---|---|---|---|---|---|---|
| R0011-E02401[Low]-1 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02401[Low]-2 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02401[Low]-3 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02401[Low]-4 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02402[Low]-1 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02402[Low]-2 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02402[Low]-3 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| R0011-E02402[Low]-4 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| J23101-E0240[Low]-1 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| J23101-E0240[Low]-2 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
| J23101-E0240[Low]-3 | 50 | 6 | 0.6 | EcoRI | 0.3 | PstI | 0.3 | 2.8 | 60 |
Electrophoresis
| M | M | M | M | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 100bp | λ | λ | 100bp | J23101-E0240[Low]-1 | J23101-E0240[Low]-2 | J23101-E0240[Low]-1 | R0011-E02401[Low]-1 | R0011-E02401[Low]-2 | R0011-E02401[Low]-3 | R0011-E02401[Low]-4 | R0011-E02402[Low]-1 | R0011-E02402[Low]-2 | R0011-E02402[Low]-3 | R0011-E02402[Low]-4 | J23101-E0240[Low]-1 | J23101-E0240[Low]-2 |
J23101-E0240[Low]-1, J23101-E0240[Low]-1, R0011-E02401[Low]-1, R0011-E02401[Low]-2, R0011-E02402[Low]-1 are inserted correctly. White colonies are not inserted <partinfo>R0011</partinfo> but its vector. Top10 we used are deleted Lac operon. Then, correctly inserted parts is green because of the lack of lacI.
Error PCR (Retry)
| Name | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | Primer VF2(10µM) | Primer VR(10µM) | Template ΔTMD failed(50ng/µL) | KOD plus ver.2 | Total |
|---|---|---|---|---|---|---|---|---|---|
| ΔTMD① | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
| ΔTMD② | 32 | 3 | 5 | 5 | 1.5 | 1.5 | 1 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 25 cycles |
| 68℃ | 4min | |
| Add DpnI 2µl | ||
| Incubate | 1h | |
| 4℃ | forever | |
Transformation
| Name | Conc(/µL) | Sample Volum(µL) | Competent Cell(µL) | Total | Plate | Incubation |
|---|---|---|---|---|---|---|
| ΔTMD① | - | 4 | 50 | 54 | LB kan | 8/6~8/9 |
| ΔTMD② | - | 4 | 50 | 54 | ||
| 2-17-F | - | 2 | 50 | 52 | ||
| 2-I-5 | 2 | 50 | 52 | LB amp |
Monday, August 9 By: Wataru, Tomonori, Ken, Takuya
Miniprep of MS and ML
| Sample number | concentration(ng/µL) |
|---|---|
| MS | 116.2 |
| ML | 146.6 |
Transfotrmation of MS and ML
| Sample | conc(ng/µL) | Sample vol(µL) | Competent Cell | Competent cell vol(µL) | Total vol(µL) | Plate | Incuvation |
|---|---|---|---|---|---|---|---|
| MS | 116.2 | 2 | KRX | 50 | 52 | LB kanamycin | 8/9 18:00‾8/10 12:00 |
| ML | 146.6 | 2 | KRX | 50 | 52 |
Restriction enzyme digestion and ethanol precipitation
To use lac p for next ligation, we digested 1-6-G by EroRI and PstI
| Sample | 10x Buffer | BSA | Enzyme (EcoRI) | Enzyme (PstI) | MilliQ | Total |
|---|---|---|---|---|---|---|
| 50 | 6 | 0.6 | 0.5 | 0.5 | 2.4 | 60 |
Incubate 37℃ 8/9 16:20‾18:20
After restriction enzyme digestion, we did ethanol precipitation.
Ligation and Transformation
| Sample | Conc (nu/µL) | Sample vol (µL) | Competent cell | Competent cell vol (µL) | Total vol (µL) | Plate | Incuvation |
|---|---|---|---|---|---|---|---|
| Lac p (low) | - | 2 | KRX | 50 | 52 | LB kanamycin | 8/9 20:00‾8/10 9:00 |
| 2 | C2 | 50 | 52 |
===Tuesday, August 10 By: Wataru, Tomonori, Ken, Fumitaka
Making culture plate on lac p (low), MS and ML
| Lac p (low) | KRX | Many colonies |
| C2 | ||
| MS | KRX | |
| ML | KRX |
Minprep of ΔTMD1+GFP
| Sample number | Concentration (ng/µL) |
|---|---|
| 1-1 | 9.9 |
| 1-2 | 27.3 |
| 2-1 | 43.2 |
| 2-2 | 34.7 |
37℃ 8/9 18:00‾8/10 9:00
Culture and Master Plate
===Wednesday, August 11 By: Wataru, Naoi, Ken, Takuya
| Sample | Medium | Cloud | Incubation |
|---|---|---|---|
| 1 | Kanamycin | o | 37℃8/10 20:00‾8/11 9:00 |
| Ampicillin | x | ||
| 2 | Kanamycin | o | |
| Ampicillin | o | ||
| 3 | Kanamycin | o | |
| Ampicillin | x | ||
| 4 | Kanamycin | o | |
| Ampicillin | x | ||
| 5 | Kanamycin | o | |
| Ampicillin | x | ||
| 6 | Kanamycin | o | |
| Ampicillin | o | ||
| 7 | Kanamycin | o | |
| Ampicillin | x |
Discussion: About sample 1, 3, 4, 5 and 7, lac promoter was correctly inserted in low copy plasmid. About sample 2 and 6, low copy plasmid and vector derived from lac promoter were ligated. We decided to use sample 1 or 3.
Miniprep of C2+lac(low), S-R-Rz 1', 3'
lac(low)1 : 31.2 (ng/µL) lac(low)2 : 29.9 (ng/µL)
Restriction Digestion and electrophoresis of lac (low) 1 and 3
| Name | EcoRI | PstI |
|---|---|---|
| 1 | 0.2 | - |
| 2 | - | 0.2 |
| 3 | 0.2 | 0.2 |
| N | - | - |
Sample: 1-1, 1-2, 1-3, 1-N, 3-1, 3-2, 3-3, 3-N
M 1-1 1-2 1-3 1-N M M 3-1 3-2 3-3 3-N M
Discussion: Each enzyme correctly cut samples.
Screening PCR of SRRz
Sample: 1-20
Control: P(1-23L) P'(2-8E) N
Maker: lambda
M N P P' P 1 2 3 4 5 6 M
7 8 9 10 11 12 13 M 14 15 16 18 19 20 M
Discussion: All of the sample were self-ligation of DT and SRRz weren't inserted.
Thursday, August 12 By: Wataru, Ken
Restriction Digestion and electrophoresis of <partinfo>B0015</partinfo>
| Sample name | Template | 10xbuffer | 100xbuffer | EcoRI | XbaI 1 | XbaI 2 | SpeI | PstI 1 | PstI 2 | Water | Total |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 3 | 1 | 0.1 | 0.2 | - | - | - | - | - | 5.7 | 10 |
| 2 | 3 | 1 | 0.1 | - | 0.2 | - | - | - | - | 5.7 | 10 |
| 3 | 3 | 1 | 0.1 | - | - | 0.2 | - | - | - | 5.7 | 10 |
| 4 | 3 | 1 | 0.1 | - | - | - | 0.2 | - | - | 5.7 | 10 |
| 5 | 3 | 1 | 0.1 | - | - | - | - | 0.2 | - | 5.7 | 10 |
| 6 | 3 | 1 | 0.1 | - | - | - | - | - | 0.2 | 5.7 | 10 |
| N | 3 | 1 | 0.1 | - | - | - | - | - | - | 5.9 | 10 |
Sample: 1-6, N Maker: lambda, 100
M 1 2 3 4 5 6 N M M M
Discussion: Each enzyme correctly cut each sample and was active.
===Thursday, August 19 By: Wataru, Tomo, Ken
Miniprep of SΔTMD1GFP
29.6(ng/µg)
Point mutation PCR of ΔTMD1GFP
| Sample number | Template | 10xbuffer | dNTPs | MgSO4 | Primer 1 | Primer 2 | Water | KOD-plus- | Total |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 31.5 | 1 | 50 |
| 2 | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 31.5 | 1 | 50 |
| control | 1.5 | 5 | 5 | 3 | 1.5 | 1.5 | 32.5 | - | 50 |
| 94(℃) | 2min | |
| 98 | 10sec | 30cycles |
| 55 | 30sec | |
| 68 | 3.5min | |
| 4.0 | forever |
Restriction Digestion(DpnI): 17:50-18:50
Electrophoresis
Sample: 1, 2, Control Marker: lambda, 100
Ligation and Transformation
We named point mutation PCR products rΔTMD1GFP.
Monday, August 23 By: Wataru, Tomo, Ken, Fumitaka, Tasuku
Miniprep of ΔTMD1
| Sample number | Concentration(ng/µg) |
| 1-1 | 58.9 |
| 2-2 | 49.9 |
Sequencing of ΔTMD1 and MS
Sample: rδTMD1GFP1-1, 2-2, and MS
Discussion: The sequencing was in success and the results were desirable. It meant point mutation of δTMD1GFP was succeeded and sequence of MS was confirmed. We decided to use rδTMD1GFP.
Screening PCR of SRRz-DT
Sample: 1-13, Marker: lambda and 100, Control:P(1-23L) and N
| 90℃ | 10min | |
| 94℃ | 30sec | 35cycles |
| 50℃ | 30sec | |
| 72℃ | 1.5min | |
| 72℃ | 4min | |
| 4℃ | hold |
M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M
Discussion: We found the band; about 200bp, and it meant the lligation was completed successfully.
Deletion PCR of rΔTMD1GFP 2-2
| Sample | 10x | dNTPs | Primer1 | Primer2 | Template | Water | KOD-plus- | Total |
|---|---|---|---|---|---|---|---|---|
| 1 | 5 | 5 | 1.5 | 1.5 | 1 | 35 | 1 | 50 |
| 2 | 5 | 5 | 1.5 | 1.5 | 1 | 35 | 1 | 50 |
| Control | 5 | 5 | 1.5 | 1.5 | 1 | 35 | - | 50 |
| 94℃ | 2min | |
| 94℃ | 10sec | 35cycles |
| 56℃ | 30sec | |
| 68℃ | 3.5min | |
| 4℃ | hold |
Restriction Digestion(DpnI)
| Template | 25(µL) |
| DpnI | 1 |
| Total | 26 |
19:10-20:10
Ligation
| Sample Template | Water | Ligation high | T4 Kinase | total | |
|---|---|---|---|---|---|
| 1 | 3 | 6 | 5 | 1 | 15 |
| 2 | 3 | 6 | 5 | 1 | 15 |
| Control | 3 | 6 | 5 | 1 | 15 |
20:15-21:15
Transformation
We named sample 1, 2 and control rrδTMD1GFP1, 2 and control.
Tuesday, August 24 By:Ken, Tomo, Tasuku, Takuya
Retry of deletion PCR of rδTMD1 GFP
| Sample | 10x | dNTPs | MgSO4 | Primer1 | Primer2 | Template | Water | KOD-plus- | Total |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
| 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50
Control||5||5||3||1.5||1.5||1||32||1||50 |
| 94℃ | 2min | |
| 94℃ | 10sec | 35cycles |
| 58℃ | 30sec | |
| 68℃ | 3.5min | |
| 4℃ | hold |
Restriction Digestion (DpnI)
14:15-15:15
Electrophoreis
Sample: 1, 2, and control, Maker: 100 and lambda M 1 2 C M
We found the band of sample 1 and 2 about 3000bp and there wasn't the band of sample control. So, we confirmed the PCR and RE were completed successfully.
Ligation
Point mutation of SRRz
| Sample | 10x | dNTPs | MgSO4 | Primer1 | Primer2 | Template | Water | KOD-plus- | total |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
| 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
| control | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 32 | 1 | 50 |
| 94℃ | 2min | |
| 98℃ | 10sec | 30cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | hold |
Restriction Digestion(DpnI), electrophoresis and ligation
We could find point mutation PCR and restriction enzyme of DpnI was done.
=PCR of E0240
| Sample | 10× | dNTPs | MgSO4 | VF2 | VR | Template | Water | KOD-plus- | Total |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31.5 | 1 | 50 |
| 2 | 5 | 5 | 3 | 1.5 | 1.5 | 1 | 31.5 | 1 | 50 |
=PCR Purification
Sample1: 5.5*50(ng/µL) Sample2: 5.2*50(ng/µL)
Restriction Digestion(EcoRI, PstI) and Gel extraction
Sample1: 28.8 (ng/µL) Sample2: 26.4 (ng/µL)
Transformation
Sample: rrΔTMD1GFP1. 2. control, and rSRRz1. 2. control
Wednesday, August 25 By:Ken, Tomo, Kazuya, Tasuku, Takuya
Making culture and Master plate
| rrΔTMD1-1 | Many Colonies |
| rrΔTMD1-2 | |
| rrΔTMD1-C- | zero |
| rSRRz-1 | Many Colonies |
| rSRRz-2 | |
| rSRRz-C- | zero |
Miniprep of 1-5G
29.0 (ng/µL)
Restriction Digestion and purification of 1-5G(low copy plasmid) and lac low
| Sample name | Template | 10xbuffer | 100xbuffer | EcoRI | SpeI | PstI | Water | Total |
|---|---|---|---|---|---|---|---|---|
| 1-5G | 50 | 6 | 0.6 | 0.4 | 0.4 | - | 2.6 | 60 |
| Lac low | 10 | 4 | 0.4 | - | 0.3 | 0.3 | 25 | 40 |
| Sample Name | Concentration(ng/µL) |
| 1-5G | 18.4 |
| Lac low | 8.6 |
Ligation of <partinfo>E0240</partinfo> and <partinfo>pSB4K5</partinfo>, Transformation
===Thursday, August 26 By:Ken, Tomo, Kazuya, Tasuku, Takuya, Fumitaka
Miniprep
| Sample name | Concentration(ng/µL) |
| constP(0.7) | 44.5 |
Restriction Digestion of constP(0.7)
| Template | 10xbuffer | 100xbuffer | SpeI | PstI | Water | Total |
|---|---|---|---|---|---|---|
| 25 | 4 | 0.4 | 0.3 | 0.3 | 10 | 40 |
Purification of constP (0.7)
49.8 ng/µL
Friday, August 27 By:Ken, Tomo, Kazuya, Fumitaka
Making master plate of E0240 low
| Sample Name | Concentration(ng/µL) |
| rrΔTMD1 1-2 | 20.9 |
| rSRRz 1-1 | 16.4 |
Restriction Digestion of rrΔTMD1 and rSRRz
| Sample name | Template | 10xbuffer | 100xbuffer | XbaI | PstI | Water | Total |
|---|---|---|---|---|---|---|---|
| rrΔTMD1 1-2 | 45 | 6 | 0.6 | 0.3 | 0.3 | 7.8 | 60 |
| rSRRz 1-1 | 45 | 6 | 0.6 | 0.3 | 0.3 | 7.8 | 60 |
(13:20-14:20)
Purification
| rrΔTMD1 1-2 | 44.7 |
| rSRRz 1-1 | 56.1 |
Lagation and transformation
lacP + rrΔTMD1 1-2 constP (0.7) + rrΔTMD1 1-2 lac low + rSRRz 1-1
Monday, August 30 By: Tomonori, Kazuya, Tasuku, Ken
Making culture and Master plate
| lacP rrΔTMD1GFP | Many colonies |
| lacP rrΔTMD1GFP(control) | Some colonies |
| constP rrΔTMD1GFP | Many colonies |
| constP rrΔTMD1GFP(control) | Many colonies |
| lacP rSRRz low | No colony |
| lacP rSRRz low(control) | No colony |
Discussion: There ware some colonies, which emitted green light, on the plate 1. So, we cultured those colonies on master plate. On the plate 5 and 6, even though we used KRX, which is able to repress lac promoter, colonies might be dead. However, we still have to do some experience so that we confirm lac promoter cannot repress enough and E. coli cannot survive.
===Tuesday, August 31 By: Tomonori, Takuya Y., Kazuya, Tasuku, Takuya, Ken
Miniprep
| constP (0.3) | 48.5 (ng/µL) |
| lac rrΔTMD1 | 107.3 |
RE of constP (0.3) and lac rrΔTMD1
Gel Extraction of lac rrΔTMD1
45min
Discussion: There were two band at the bottom of the gel. It was too long -45min-, and insert and vector might be contaminated. But we went on next operation.
Purification of constP (0.3) and lac rrΔTMD1
| constP (0.3) | 5.8 (ng/µL) |
| lac rrΔTMD1 | 7.8 (ng/µL) |
Ligation and transformation
| Insert | Vector |
| lac rrΔTMD1 | constP (0.3) |
===Wednesday, September 1 By: Tomonori, Kazuya, Tasuku, Fumitaka, Ken
Making culture and Master plate
| lac rrΔTMD1 constP | many colonies |
| lac rrΔTMD1 const (control) | many colonies |
Screenig PCR of lacP-rrΔTMD1GFP-constP Sample: 1-13 Control: Positive (1-23L) Maker: lambda, 100
M 1 2 3 4 5 6 7 8 9 10 11 12 13 P M
Discussion: All of the sample except sample 10 might be self-ligation products of constP.
Miniprep
| rSRRz 1-1 | 33.8 (ng/µL) |
| low | 56.0 (ng/µL) |
Restriction Digestion of rSRRz and low
| Sample name | Template | 10xbuffer | 100xbuffer | EcoRI | PstI | Water | Total |
|---|---|---|---|---|---|---|---|
| rSRRz | 20 | 4 | 0.4 | 0.3 | 0.3 | 15 | 40 |
| low | 20 | 4 | 0.4 | 0.3 | 0.3 | 15 | 40 |
(13:25-14:30)
Purification
| rSRRz | 6.5 (ng/µL) |
| low | 16.8 |
Ligation and transformation
Insert: rSRRz 1-1 Vector: low copy plasmid
Thursday, September 2 By: Tomonori, Tomo, Takuya, Ken
Making culture and Master plate
| rSRRz low | 13 colonies |
| rSRRz low (Control) | 13colonies |
Screening PCR of rSRRz low
Sample: rSRRz (1‾13) Maker: lambda, 100 Control: Positive (1-23L), Neganive
M 1 2 3 4 5 6 7 8 9 10 11 12 13 P N M
Discussion: From sample 1, two vectors might be ligated. Sample 3 and 4, rSRRz might be inserted in low copy plasmid correctly. Sample 11, it might be the self-ligation product of low copy plasmid. Anyway, we decided to culture those 4 colonies on master plate.
Friday, September 3 By: Tomonori, Tomo, Kazuya, Tasuku, Fumitaka, Ken
Making culture
lac rrΔTMD1 1, 3 rrΔTMD1 1-1, 1-2 rSRRz 1-1, 1-2 ML
