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JULY: WEEK 3
July, 12th
Phasins PhaP1 and PhaP2 were sequenced, but none of them has a clear chromatogram, so we decided to sequence the PCR results.
 Gel run/cut of phasins amplified via PCR Phasins were gel run/cut and the samples were prepared to be sequenced.
Inoculum of
- <partinfo>BBa_J23101</partinfo>
- <partinfo>BBa_J23105</partinfo>
- <partinfo>BBa_J23106</partinfo>
for tomorrow ligations.
July, 13th
LB+Amp was prepared and phasins sample were sent to be sequenced.
For cultures grown OverNight at 37°C, 220 rpm were MiniPrep was performed:
| <partinfo>BBa_J23101</partinfo> | XXX ng/ul
|
| <partinfo>BBa_J23105</partinfo> | XXX ng/ul
|
| <partinfo>BBa_J23106</partinfo> | XXX ng/ul
|
Other plasmids were retrieved from our freezer:
- <partinfo>BBa_J23118</partinfo> (already digested SpeI-PstI)
- <partinfo>BBa_J23110</partinfo> (already digested SpeI-PstI)
- <partinfo>BBa_J23116</partinfo> (already digested SpeI-PstI)
- I6-2 (already digested XbaI-PstI)
- 4C5 (MiniPrep performed, it will be digested EcoRI-PstI)
- I7-3 (MiniPrep performed, it will be digested EcoRI-PstI)
- I8-5 (MiniPrep performed, it will be digested EcoRI-PstI)
- I10-1 (MiniPrep performed, it will be digested EcoRI-PstI)
- I3-1 (MiniPrep performed, it will be digested XbaI-PstI)
Digestion of:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 | Enzyme 2 | Buffer H
|
| 4C5 (x2) | Vector | 25 | 3,6 | 16,9 | 1 EcoRI | 1 PstI | 2,5
|
| I7-3 | Insert | 25 | 14 | 6,5 | 1 EcoRI | 1 PstI | 2,5
|
| I8-5 | Insert | 25 | 12,7 | 7,8 | 1 EcoRI | 1 PstI | 2,5
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| I10-1 | Insert | 25 | 15,3 | 5,2 | 1 EcoRI | 1 PstI | 2,5
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| I3-1 (x2) | Vector | 25 | 5,8 | 14,7 | 1 XbaI | 1 PstI | 2,5
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| <partinfo>BBa_J23105</partinfo> | Insert | 25 | 15,9 | 4,6 | 1SpeI | 1 PstI | 2,5
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| <partinfo>BBa_J23106</partinfo> | Insert | 25 | 13 | 7,5 | 1 SpeI | 1 PstI | 2,5
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| <partinfo>BBa_J23101</partinfo> | Insert | 25 | 10,4 | 10,1 | 1 SpeI | 1 PstI | 2,5
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Digestions were incubated at 37°C for 3hours, then gel run/cut.
Gel was prepared for electrophoresis: 150ml TBE + 1,5 g Agarose + 3ul EtBr.
Ligations were all performed 1:5 (1ul vector + 5ul insert):
- I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
- I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
- I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
- I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
- I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
- I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
- I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
- I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
- I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
- I74C5= I7 (E-P) + 4C5 (E-P)
- I84C5= I8 (E-P) + 4C5 (E-P)
- I104C5= I10 (E-P) + 4C5 (E-P)
July, 14th
Trasformation of ligations in:
| Ligation name | E. coli strain | Resistance |
|---|
- I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
|
TOP10
|
Amp 100 |
- I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
|
TOP10
|
Amp 100 |
- I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
|
TOP10
|
Amp 100 |
- I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
|
DH5alpha
|
- I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
|
DH5alpha
|
Amp 100 |
- I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
|
DH5alpha
|
Amp 100 |
- I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
|
DH5alpha
|
Amp 100 |
- I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
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DH5alpha
|
Amp 100 |
- I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
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DH5alpha
|
Amp 100 |
- I74C5= I7 (E-P) + 4C5 (E-P)
|
TOP10
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Cm 12.5 |
- I84C5= I8 (E-P) + 4C5 (E-P)
|
TOP10
|
Cm 12.5 |
- I104C5= I10 (E-P) + 4C5 (E-P)
|
TOP10
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Cm 12.5 |
 Quality control for HELPER and CRIM plasmids July, 15th
 Quality control for pBHR68 and CRIM plasmids July, 16th
 Screening ligations I11-I16 |  Screening ligations I17-I19, I7/I8/I10-4C5 |
Since it's impossible to isolate the CRIM plasmid <partinfo>BBa_J72007</partinfo> we created a pseudo CRIM plasmid to check that not pir strains aren't able to propagate it.
We called it "THE RING" (RING) and we built it from our I5 (Cm resistance - <partinfo>BBa_P1004</partinfo> - + TT - <partinfo>BBa_B0015</partinfo> - + R6K ori - <partinfo>BBa_J61001</partinfo>) in <partinfo>pSB1A2</partinfo>:
- Digestion of I5 XbaI-PstI
- Gel extraction
- Quantification with Nanodrop (15ng/ul)
- Ligation of X and P sites each other (used 2ul of DNA)
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