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Team:Heidelberg/Tyrocidine week21 interms
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After amplification, an analytical gel electrophoresis was run to quantify DNA concentrations. | After amplification, an analytical gel electrophoresis was run to quantify DNA concentrations. | ||
| - | [[File: | + | [[File:Heidelberg_20130917_2log_bb_ccdB_C(TycC2)_C(TycC2)Q5_6B_15_QUANT_caption.png|thumb|right|100px| Check on PCR-products by gel eletrophoresis. All products showed an appropriate size.]] |
:'''C(TycC2) PCR contents''' | :'''C(TycC2) PCR contents''' | ||
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After 10 hours of culture growth, colony PCRs and test digests with Pst1 and EcoRI were conducted. Afterwards, minipreps of cultures containing the plasmids and concentration measurements were performed accompanied by test digests. Finally, positive constructs were transformed again and prepared for sequencing. | After 10 hours of culture growth, colony PCRs and test digests with Pst1 and EcoRI were conducted. Afterwards, minipreps of cultures containing the plasmids and concentration measurements were performed accompanied by test digests. Finally, positive constructs were transformed again and prepared for sequencing. | ||
| - | + | ||
| - | File: 20130918_COLPCR_ccdB-Ind_labeled.png|Colony PCRs. On the left: ccdB (all positive except 8). On the right: Indigoidine (all positive). | + | [[File:Heidelberg_ 20130918_COLPCR_ccdB-Ind_labeled.png| 150px |Colony PCRs. On the left: ccdB (all positive except 8). On the right: Indigoidine (all positive).]] |
| - | File: 20130919_1kb_ccdb-indc1-5_digest_labeled.png|Digests of ccdB-indC constructs 1-5 with Pst1 and EcoRI. | + | [[File:Heidelberg_ 20130919_1kb_ccdb-indc1-5_digest_labeled.png| 150px |Digests of ccdB-indC constructs 1-5 with Pst1 and EcoRI.]] |
| - | File: 20130919_1kb_ccdbIndc_constructs5-6_digest_labeled.png| Digests of ccdB-indC constructs 6-10 with Pst1 and EcoRI. | + | [[File:Heidelberg_ 20130919_1kb_ccdbIndc_constructs5-6_digest_labeled.png| 150px | Digests of ccdB-indC constructs 6-10 with Pst1 and EcoRI.]] |
| - | File: 20130919_1kb_digest_ccdbind_labeled.png|Digests of ccdB-indC3 and 7 with Pst1 and EcoRI. Results were positive. | + | [[File:Heidelberg_ 20130919_1kb_digest_ccdbind_labeled.png| 150px |Digests of ccdB-indC3 and 7 with Pst1 and EcoRI. Results were positive.]] |
| - | + | ||
:'''ccdB nanodrop measurements''' | :'''ccdB nanodrop measurements''' | ||
Latest revision as of 19:30, 4 October 2013
ccdb Construct
Amplification
ccdb-C(tycC2)-indC plasmid (pJS01) Amplification of C(tycC2), ccdB and pSB1C3 DNA fragments (indC was stored in the fridge from former experiments).
- ccdB pcr contents
| What | µl |
|---|---|
| primer_fw (PW38) | 2 |
| primer_rv (PW41) | 2 |
| template (Gateway pDONR vector) | 2 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 4 |
- PCR-Program ccdB sequence
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 0:10 |
| 8 | 98 | 0:01 |
| 70 ↓0.5 | 0:05 | |
| 72 | 0:15 | |
| 28 | 98 | 0:01 |
| 65 | 0:05 | |
| 72 | 0:15 | |
| 1 | 72 | 1:30 |
| 1 | 10 | inf |
- pSB1C3 PCR contents
| wWhat | µl |
|---|---|
| primer_fw (PW23) | 2 |
| primer_rv (PW42) | 2 |
| template (pSB1C3) | 0.5 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 5.5 |
- PCR-Program pSB1C3 sequence
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 2:00 |
| 35 | 98 | 0:05 |
| 60 | 0:20 | |
| 72 | 1:00 | |
| 1 | 72 | 10:00 |
| 1 | 10 | inf |
- C(TycC2) PCR contents
| What | µl |
|---|---|
| primer_fw(PW21) | 2 |
| primer_rv(PW14) | 2 |
| template (B. Parabrevis) | 1 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 5 |
- PCR-Program C(TycC2)
| Cycles | Temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 2:00 |
| 35 | 98 | 0:01 |
| 59 | 0:05 | |
| 72 | 1:00 | |
| 1 | 72 | 5:00 |
| 1 | 10 | inf |
After amplification, an analytical gel electrophoresis was run to quantify DNA concentrations.
_C(TycC2)Q5_6B_15_QUANT_caption.png)
- C(TycC2) PCR contents
| What | Size [bp] | Estimated concentration |
|---|---|---|
| pSB1C3 | 2500 | 35 |
| ccdB | 600 | 30 |
| C(TycC2) | 1000 | 30 |
| indC | 4000 | 100 |
Assembly
Gibson assembly was followed by purification with isopropanol and transformation by heat shock into ccdB resistent and non-resistent (Top10) cells.
After 10 hours of culture growth, colony PCRs and test digests with Pst1 and EcoRI were conducted. Afterwards, minipreps of cultures containing the plasmids and concentration measurements were performed accompanied by test digests. Finally, positive constructs were transformed again and prepared for sequencing.
- ccdB nanodrop measurements
| Colony | Concentration [ng/µl] |
|---|---|
| ccdB1 | 863.5 |
| ccdB2 | 436.9 |
| ccdB3 | 1258.5 |
| ccdB4 | 260.5 |
| ccdB5 | 106.6 |
| ccdB6 | 1064.4 |
| ccdB7 | 1213.0 |
| ccdB8 | 1301.4 |
| 3B | 982.3 |
| 3*B | 982.4 |
Sequencing results of ccdB3 and ccdB7 (just partwise sequencing) were positive. Biobrick can be submitted.
