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Team:Warsaw/Calendar-Stage1/12 July 2010
From 2010.igem.org
(Difference between revisions)
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Monday, 12 June 2010 | Monday, 12 June 2010 | ||
| - | <div class="note">Cloning GFP+terminator behind Anderson's RBSes </div> | + | <br><br><div class="note">Cloning GFP+terminator behind Anderson's RBSes </div> |
| - | + | ||
| - | + | 1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1 | |
<br>2. Digest samples 2.1, 3.1, 4.1, 5.1, 6.1 (Anderson's RBSes+GFPterminator) with EcoRI +PstI to examine the correctness of obtained constructs. | <br>2. Digest samples 2.1, 3.1, 4.1, 5.1, 6.1 (Anderson's RBSes+GFPterminator) with EcoRI +PstI to examine the correctness of obtained constructs. | ||
<br>3. Gel electrophoresis.[Gel 1] | <br>3. Gel electrophoresis.[Gel 1] | ||
<br>4. All constructs are of the expected size. | <br>4. All constructs are of the expected size. | ||
| - | <br> | + | |
| - | <div class="note">Cloning Connumity RBSes+GFP+terminator behind 23100 </div> | + | <br><br><div class="note">Cloning Connumity RBSes+GFP+terminator behind 23100 </div> |
| - | + | 1. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI, | |
<br>2. Gel electrophoresis [Gel 3] | <br>2. Gel electrophoresis [Gel 3] | ||
| - | <br>3. Digest community RBSes-GFP-terminator, samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F with XbaI + PstI | + | <br>3. Digest community RBSes-GFP-terminator, samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F with XbaI + PstI |
<br>4. Gel electrophoresis [Gel 2, Gel 3] | <br>4. Gel electrophoresis [Gel 2, Gel 3] | ||
<br>5. Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F | <br>5. Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F | ||
| - | <br> | + | <br><br><div class="note">Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100</div> |
| - | <div class="note">Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100</div> | + | 1. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS) with SpeI and PstI |
| - | + | ||
| - | + | ||
| - | + | ||
<br>2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins: | <br>2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins: | ||
<br>- GFP E240 | <br>- GFP E240 | ||
| Line 38: | Line 35: | ||
<br>- mCherry J67102* | <br>- mCherry J67102* | ||
<br>- mCherry NLS J130012 | <br>- mCherry NLS J130012 | ||
| - | |||
<br>* can have an internal RBS. Check by cloning behind J23100. | <br>* can have an internal RBS. Check by cloning behind J23100. | ||
<br>3. Gel electrophoresis [Gel 4] | <br>3. Gel electrophoresis [Gel 4] | ||
<br>4. Gel extraction and ligation of fluorescent proteins with expression vector. | <br>4. Gel extraction and ligation of fluorescent proteins with expression vector. | ||
| - | |||
<br>______________________________________________ | <br>______________________________________________ | ||
<br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek): | <br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek): | ||
| Line 67: | Line 62: | ||
<br> | <br> | ||
<div class="note"> GEL 2</div> | <div class="note"> GEL 2</div> | ||
| - | + | Samples: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator] | |
<br>RBSes+FPterminator at expected size, vectors at expected size; | <br>RBSes+FPterminator at expected size, vectors at expected size; | ||
<img src="https://static.igem.org/mediawiki/igem.org/d/de/12_07_communityRBS_Xba_Pst.JPG"/> | <img src="https://static.igem.org/mediawiki/igem.org/d/de/12_07_communityRBS_Xba_Pst.JPG"/> | ||
<div class="note"> GEL 4</div> | <div class="note"> GEL 4</div> | ||
| - | + | Samples: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012 | |
<br>Not all samples at expected size, some vectors at wrong size. | <br>Not all samples at expected size, some vectors at wrong size. | ||
<br>Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size. | <br>Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size. | ||
Revision as of 20:31, 13 July 2010
Monday, 12 June 2010
2. Digest samples 2.1, 3.1, 4.1, 5.1, 6.1 (Anderson's RBSes+GFPterminator) with EcoRI +PstI to examine the correctness of obtained constructs.
3. Gel electrophoresis.[Gel 1]
4. All constructs are of the expected size.
2. Gel electrophoresis [Gel 3]
3. Digest community RBSes-GFP-terminator, samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F with XbaI + PstI
4. Gel electrophoresis [Gel 2, Gel 3]
5. Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F
2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
- GFP E240
- CFP E0420
- YFP E0430
- mOrange E2050*
- mCherry J67102*
- mCherry NLS J130012
* can have an internal RBS. Check by cloning behind J23100.
3. Gel electrophoresis [Gel 4]
4. Gel extraction and ligation of fluorescent proteins with expression vector.
______________________________________________
Electrophoresis (opis zdjęć wg kolejności naniesionych próbek): Gel 1: 2.1, 3.1, 4.1, 5.1, 6.1 [Anderson RBSes+GFPterminator] Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator] Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100] Gel 4: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
______________________________________________
Digest conditions, used for all reactions:
8 μl DNA
2 μl BamHI buffer
10 μl H2O
0,3 μl enzymów
20 μl final volume
digest for 3h
Ligation conditions, used for all reactions:
25ul insert DNA
2ul vector DNA
3ul ligase buffer
30 ul final volume
overnight ligation
RBSes+FPterminator at expected size, vectors at expected size;
Not all samples at expected size, some vectors at wrong size.
Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size.
Cloning GFP+terminator behind Anderson's RBSes
1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1
2. Digest samples 2.1, 3.1, 4.1, 5.1, 6.1 (Anderson's RBSes+GFPterminator) with EcoRI +PstI to examine the correctness of obtained constructs.
3. Gel electrophoresis.[Gel 1]
4. All constructs are of the expected size.
Cloning Connumity RBSes+GFP+terminator behind 23100
1. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,
2. Gel electrophoresis [Gel 3]
3. Digest community RBSes-GFP-terminator, samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F with XbaI + PstI
4. Gel electrophoresis [Gel 2, Gel 3]
5. Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F
Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100
1. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS) with SpeI and PstI
2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
- GFP E240
- CFP E0420
- YFP E0430
- mOrange E2050*
- mCherry J67102*
- mCherry NLS J130012
* can have an internal RBS. Check by cloning behind J23100.
3. Gel electrophoresis [Gel 4]
4. Gel extraction and ligation of fluorescent proteins with expression vector.
______________________________________________
Electrophoresis (opis zdjęć wg kolejności naniesionych próbek): Gel 1: 2.1, 3.1, 4.1, 5.1, 6.1 [Anderson RBSes+GFPterminator] Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator] Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100] Gel 4: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
______________________________________________
Digest conditions, used for all reactions:
8 μl DNA
2 μl BamHI buffer
10 μl H2O
0,3 μl enzymów
20 μl final volume
digest for 3h
Ligation conditions, used for all reactions:
25ul insert DNA
2ul vector DNA
3ul ligase buffer
30 ul final volume
overnight ligation
GEL 2
Samples: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
RBSes+FPterminator at expected size, vectors at expected size;
GEL 4
Samples: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
Not all samples at expected size, some vectors at wrong size.
Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size.
