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July 1st
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July 2nd
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July 3rd
-
Fim Switch
- Marcia + Lais
-
Digest ran for K1632013 (MP17) and K137058 (MP18)
37C 1 hour, 64C 20 minutes, 4C rest
2 copies of each sample digested
17 + 18 - Arac/FimE
22 + 21 - FimS/GFP
For ligation, samples were quantifies on the nanodrop
MP17 - 264.2 ng/µl
MP18 - 306.8 ng/µl
MP22 - 143.9 ng/µl
MP21 - 117.5 ng/µl
Competent cell test kit Calculating cell efficiency
Ran gel + cut bands
MP17 - 0.193 g
MP18 - 0.205 g
MP21 - 0.202 g
MP22 - 0.292 g
Gel purified using the Qiagen purification kit
Competent cells testing kit using the iGEM protocol
Controls-
DH5α + H20 in LB
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DH5α + H20 on cm
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DH5α + GFP on cm
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DH5α + mRFP on amp
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DH5α + H20 in LB
July 4th
-
Fim Switch
- Ansh + Lais
-
Counting cell efficiency
---10 50 100
1 - 2 20 46
2 - 9 37 73
3 - 6 36 34
- Lais, Ansh, Marcia + Sophie
- Transformation
July 5th
- Description
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July 6th
- Marcia, Sophie + Ansh
-
Digestion
MP17
• 2 µl buffer (cutsmart)
• 3.8 µl DNA
• 1 µl Pst1
• 1 µl Spe1
• 12.2 µl water
MP21
• 2 µl buffer (cutsmart)
• 8.5 µl DNA
• 1 µl Pst1
• 1 µl Xba1
• 7.5 µl water
Incubate at 37C 1 hour, 64C 20 minutes, 4C rest
July 7th
- Sophie + Marcia
- Used E-Gel to purify digest
20 µl of MP17 and MP21, no dye added
25 µl water in empty wells
1kb plus ladder - 10 µl in M lane
MP17 in lane 4
MP21 in lane 5
Digestion wasn't successful, MP21 was smeared in the gel
Extracted MP17 but not MP21
July 8th
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July 9th
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July 10th
-
Fim Switch
- Lais, Marcia + Sophie
-
Digestion
Digested MP18 and MP22
Ran gel after digest and cut the band
MP18 - 0.255 g
MP22 - 0.274 g
Gel extraction using Qiagen extraction kit
Ligation
Ligating at 1:1, 1:3 and 1:5 ratios
1:1
• 2 µl buffer x10
• 1 µl vector (MP18)
• 1 µl insert (MP22)
• 1 µl T4 ligase
• 15 µl water
1:3
• 2 µl buffer x10
• 1 µl vector (MP18)
• 3 µl insert (MP22)
• 1 µl T4 ligase
• 13 µl water
1:5
• 2 µl buffer x10
• 1 µl vector (MP18)
• 5 µl insert (MP22)
• 1 µl T4 ligase
• 11 µl water
Set overnight (16 h) at 16C
July 11th
-
Fim Switch
- Lais, Marcia + Sophie
- Transformed cells
Used Bradley's protocol
July 12th
-
Fim Switch
- Whats this one?
- 10 ml -> LB final volume
CAM 2.5 mg/ml -> 12.5 µg/mg
µg 12.5 x 10 = 2500 x V
V = 0.05 ml
= 50 µl
10 ml -> LB
50 µl -> Anti B -> CM
1 colony
Colony from 1:3 plate transformation started
July 13th
Fim Switch
- Marcia + Lais
- Miniprepped Bradley's stuff, FimE and Lais' promoters
Following Qiagen protocol
July 14th
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July 15th
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July 16th
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July 17th
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July 18th
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July 19th
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July 20th
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July 21st
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July 22nd
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July 23rd
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July 24th
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July 25th
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July 26th
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July 27th
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July 28th
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July 29th
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July 30th
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