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April
1st
Organization
Getting all kind of lab equipment
- key cards and keys for the lab
- cooled centifuge
- space reservation in -80°C freezer
- bottles for 70% ethanol
- ... (problems of a first year team ^^)
12th
- chemically competent E. coli NEB Top10, DH5α and BL21 were made
15th
- 800 ml LB for plates with 1.5% agar and kanamycin (50 µg/L) were prepared
- test of transformation efficiency of our competent cells
- Colonies on the agar plates were counted after transformation with 1 µl DNA (147 ng/µl)
- Cells had been incubated in SOC or LB medium
| Dilution | 200 µl (stock) | 100 µl (stock) | 1:5 | 1:10 | 1:100
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| DH5α | SOC: 30 LB: 10 | SOC: 7 LB: 1 | SOC: 1 LB: 2 | SOC: 2 LB: 1 |
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| NEB 10β | SOC: 170 LB: 135 | SOC: 100 LB: 79 | SOC: 25 LB: 22 | SOC: 9 LB: 9 | SOC: 1 LB:0
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- BL21 cells were centrifuged and plated; only 128 colonies grew in total indicating a very bad efficiency (~482.11).
Following formula was used to calculate the efficiency: Efficiency = (CFU /µg DNA) / Dilution
- for DH5α: medial efficiency in SOC: 1864.36, and in LB: 440.68
- for NEB: medial efficiency in SOC: 6779.66, and in LB: 4698.30
- Higher efficiency when using SOC!
22th
Pcq lab PCR advanced primus 25, 96
| | Lenght [bp] | % GC | Tm | TA
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| EYFP_RFC25 | 778 | 61 | 84 | 56
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| REACh1_C | 481 | 62 | 84 | 57
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| REACh1_N | 320 | 59 | 82 | 54
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| REACh2_C | 487 | 62 | 83 | 57
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| REACh2_N | 320 | 59 | 82 | 54
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| Parameter | Duration | Temp [°C]
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| Denature | 10:00 | 98
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| Denature | 00:30 | 98
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| Anneal | 00:30 | 52
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| Elongate | 02:36 | 72
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| Elongate | 05:00 | 72
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| Store | forever | 8
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- RFC25_F RFC25_R
- RFC25_F REACh1_R
- REACh1_F RFC25_R
- RFC25_F REACh2_R
- REACh2_F RFC25_R
23th
- 5 µl of the PCR products were run on a 1,5% agarose gel
- DNA bands were purified from gel with the High Pure Product Purification Kit. Full length products were contaminated with REACh1
| | 20x | -> 20 µL thereof: 30x
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| HF | 10 | 10
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| dNTP | 4 | 4
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| Template | 2x 1 | 10
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| Phusion | 0.5 | 0.5
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| Primer | - | 2x 2.5
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| Water | 33.5 | 21.5
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Anneling: 52°C
Elongation: 20 sec
- PCR with Phusion polymerase
| | Volume [µL]
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| HF | 10
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| dNTP | 4
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| Template | 1
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| Phusion | 0.5
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| Primer | 2x 2.5
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| Water | 29.5
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| Total | 50
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- Digestion for restriction with NEB enzymes
| | Volume
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| Destination Vector | 500 ng
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| EcoRI-HF | 1 µL
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| PstI | 1 µL
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| 10x NEB Buffer 2.1 | 5 µL
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| Water | ??? µL
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| Total | ??? µL
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28th
- The DNA concentration of the SOE2 products were measured after purification
- 1: 57 ng/µL
- 2: 69.5 ng/µL
29th
30th
- A PAGE of the SOE2 products was run on agarose gel for checking
- → no positive results; only full length products were amplified
- SOE PCRs were run again
- → more template
- → hot start
- → faster
- → elongation: 15 min
- → 20 cycles
Template A+B → 20 µL → 13.8 + 6.2 5.3 + 14.7
| | volume [µL]
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| HF | 10
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| dNTP | 4
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| Template | 20
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| Phusion | 0.5
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| Water | 13.5
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