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Team:Heidelberg/Templates/DelH week5
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(Created page with " == 27-05 - 02-06-13 == ===Generation of pSB6A1 === ====Miniprep==== * Grow pSB6A1 from glycerol stock in 2 ml LB Amp ON at 37°C. * Miniprep using miniprep kit from Qiagen and d...") |
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| - | == 27-05 - 02-06-13 == | + | ==27-05 - 02-06-13 == |
===Generation of pSB6A1 === | ===Generation of pSB6A1 === | ||
====Miniprep==== | ====Miniprep==== | ||
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Expected band: 3985 bp (pSB6A1) and at 1106 bp (insert=J04450) | Expected band: 3985 bp (pSB6A1) and at 1106 bp (insert=J04450) | ||
<br/> | <br/> | ||
| - | [[File: | + | [[File:Heidelberg_20130528 2log 2xpSB6A1.png|200px|thumb|right|'''Fig.5.1''' Generation of pSB6A1 for Backbone generation (loaded 50 µL of PCR) <br> ''l1:''2-log ladder, ''l2-3:''digested pSB6A1 <br/> expected band at 4 Kb was cut out]] |
| - | + | ||
:=> Expected bands are present. Backbone pSB6A1 was cut out and gel purified (c=4 ng/µl). | :=> Expected bands are present. Backbone pSB6A1 was cut out and gel purified (c=4 ng/µl). | ||
| + | <div style="clear:both"></div> | ||
===Generation of AraC Fragment=== | ===Generation of AraC Fragment=== | ||
====Gel Extraction==== | ====Gel Extraction==== | ||
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====Result==== | ====Result==== | ||
2 of the colonies turned blue | 2 of the colonies turned blue | ||
| - | :=> Perform miniprep. | + | :=> Perform [https://2013.igem.org/Team:Heidelberg/Project/Methods miniprep]. |
| + | |||
====Miniprep==== | ====Miniprep==== | ||
Miniprep was performed according to Qiagen's instructions. | Miniprep was performed according to Qiagen's instructions. | ||
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Expected band: 5 Kb | Expected band: 5 Kb | ||
<br/> | <br/> | ||
| - | [[File: | + | [[File:Heidelberg_20130527 2log 1a 1b 2log 2.png|200px|thumb|right|'''Fig.5.2''' PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR) <br> ''l1:''2-log ladder, ''l3-4:''F1a,''l5-6:''F1b,''l7-8:''F2 ]] |
| - | + | ||
The gel shows a lot unspecific bands for both DelH F1a samples. | The gel shows a lot unspecific bands for both DelH F1a samples. | ||
:=> Fragment was cut and gel purified. | :=> Fragment was cut and gel purified. | ||
<br/> | <br/> | ||
| - | + | <div style="clear:both"></div> | |
=== DelH F1b=== | === DelH F1b=== | ||
====PCR Conditions F1b.W5.A==== | ====PCR Conditions F1b.W5.A==== | ||
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Expected band: 5 Kb | Expected band: 5 Kb | ||
<br/> | <br/> | ||
| - | [[File: | + | [[File:Heidelberg_20130527 2log 1a 1b 2log 2.png|200px|thumb|right|'''Fig.5.2''' PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR) <br> ''l1:''2-log ladder, ''l3-4:''F1a,''l5-6:''F1b,''l7-8:''F2 ]] |
| - | + | ||
Gel shows one band at the expected length of 5 Kb, but also other side bands below and a huge, bright band at 2 Kb. | Gel shows one band at the expected length of 5 Kb, but also other side bands below and a huge, bright band at 2 Kb. | ||
:=> Band was cut and gel isolated. | :=> Band was cut and gel isolated. | ||
<br/> | <br/> | ||
| + | <div style="clear:both"></div> | ||
=== DelH F2=== | === DelH F2=== | ||
====PCR Conditions F2.W5.A==== | ====PCR Conditions F2.W5.A==== | ||
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Expected length: 8 Kb | Expected length: 8 Kb | ||
<br/> | <br/> | ||
| - | [[File: | + | [[File:Heidelberg_20130527 2log 1a 1b 2log 2.png|200px|thumb|right|'''Fig.5.2''' PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR) <br> ''l1:''2-log ladder, ''l3-4:''F1a,''l5-6:''F1b,''l7-8:''F2 ]] |
| - | + | ||
There was a specific band at 8 Kb. | There was a specific band at 8 Kb. | ||
:=> Fragment was cut and gel extracted. | :=> Fragment was cut and gel extracted. | ||
<br/> | <br/> | ||
| + | <div style="clear:both"></div> | ||
Latest revision as of 18:47, 23 October 2013
Contents |
27-05 - 02-06-13
Generation of pSB6A1
Miniprep
- Grow pSB6A1 from glycerol stock in 2 ml LB Amp ON at 37°C.
- Miniprep using miniprep kit from Qiagen and dilute in 30 µl ddH2O.
Result
Concentration 69.5 ng/µl
Digest
- The pSB6A1 was generated from the parts registry and cut with EcoRI & PstI
| Template DNA | BSA | NEBuffer2 | Enzymes | ddH2O | Total amount |
|---|---|---|---|---|---|
| 15 µl (69.5 ng/µl) | 5 µl | 5 µl | 2x 1.5 µl EcoRI-HF & PstI | 22 µl | 50 µl |
- The digest was incubated 45 min at 37°C.
Result
Expected band: 3985 bp (pSB6A1) and at 1106 bp (insert=J04450)
l1:2-log ladder, l2-3:digested pSB6A1
expected band at 4 Kb was cut out
- => Expected bands are present. Backbone pSB6A1 was cut out and gel purified (c=4 ng/µl).
Generation of AraC Fragment
Gel Extraction
Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.
Result
The measured concentrations are shown in the table below and was stored at -20°C.
| Sample | Concentration [ng/µl] |
|---|---|
| AraC | 22.5 |
Generation of lacZ Fragment
Gel Extraction
Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.
Result
The measured concentrations are shown in the table below and was stored at -20°C.
| Sample | Concentration [ng/µl] |
|---|---|
| lacZ | 42.5 |
Generation of Backbone pSB6A1-AraC-lacZ
Ligation of pSB6A1 with AraC-lacZ
A ligation was performed of fragments for 1 h at RT.
| Sample | Concentration [ng/µl] | Amount for ligation [µl] |
|---|---|---|
| pSB6A1 | 4 | 12.5 |
| AraC-lacZ | 22.5 | 3.5 |
| T4-ligase | - | 1 |
| Buffer | - | 2 |
| ddH2O | - | 1 |
| Final volume | - | 20 |
- The ligase was inactivated by heat shock for 5 min at 70°C.
Transformation
- Afterwards, a transformation was performed with 15 µl of the ligation reaction in competent TOP10.
- Cells were streaked on LB Amp plates and incubated ON at 37°C.
Induction
- The following day, 5 white colonies were picked (because the red ones still have the mRFP insert J04450) and were incubated in 2 ml LB Amp with 20 µl Arabinose (10%) and 100 µl X-Gal (20 ng/µl).
Result
2 of the colonies turned blue
- => Perform miniprep.
Miniprep
Miniprep was performed according to Qiagen's instructions.
Result
| Sample | Concentration [ng/µl] |
|---|---|
| pSB6A1-AraC-lacZ | 99 |
DelH F1a
PCR Conditions F1a.W5.A
| Reagent | DelH F1a | DelH F1a |
|---|---|---|
| Expected length [Kb] | 5 | 5 |
| Template | 1 µl D. acidovorans | 1 µl D. acidovorans |
| Primer fw 10 µM | DN01 | DN01 |
| Primer rev 10 µM | DN08 | DN08 |
| Phusion Master Mix (2x) | 25 µl | 25 µl |
| DMSO | 2.5 µl | - |
| ddH2O | 20,5 | 23 µl |
| Cycles | Temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 10 |
| 16 | 98 | 1 |
| 62 | 5 | |
| 72 | 2:00 min | |
| 1 | 72 | 2:30 min |
| 1 | 4 | inf |
Result
Expected band: 5 Kb
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2
The gel shows a lot unspecific bands for both DelH F1a samples.
- => Fragment was cut and gel purified.
DelH F1b
PCR Conditions F1b.W5.A
| Reagent | DelH F1b |
|---|---|
| Expected length [Kb] | 5 |
| Template | 1 µl D. acidovorans |
| Primer fw 10 µM | DN07 |
| Primer rev 10 µM | DN02 |
| Phusion Master Mix (2x) | 25 µl |
| DMSO | - |
| ddH2O | 23 µl |
| Cycles | Temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 10 |
| 16 | 98 | 1 |
| 68 | 5 | |
| 72 | 2:00 min | |
| 1 | 72 | 2:30 min |
| 1 | 4 | inf |
Result
Expected band: 5 Kb
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2
Gel shows one band at the expected length of 5 Kb, but also other side bands below and a huge, bright band at 2 Kb.
- => Band was cut and gel isolated.
DelH F2
PCR Conditions F2.W5.A
| Reagent | DelH F2 |
|---|---|
| Expected length [Kb] | 8 |
| Template | 1 µl glycerol stock |
| Primer 10 µM fw | 0.5 µl DelH_f2_SalI_fw |
| Primer 10 µM rev | 0.5 µl DelH_f2_KpnI_rev |
| Phusion Master Mix (2x) | 25 µl |
| DMSO | 0 µl |
| ddH2O | 23 µl |
| Cycles | Temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 66 | 5 | |
| 72 | 2:45 min | |
| 1 | 72 | 2:45 min |
| 1 | 4 | inf |
Result
Expected length: 8 Kb
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2
There was a specific band at 8 Kb.
- => Fragment was cut and gel extracted.
