Team:Goettingen/NoteBook w7

Transformation from 15.7.13

-          Amp-plates:

negative control = negative/no clones

transformation of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate

-          Cm-plates

Negative control = negative

Clones on all other plates including w/o insert control (forgot gel extraction of pSB1C3 vector à terminator insert is still in reaction mix; since we didn’t dephosphorylate the vector with AP, there will be false positives corresponding to plasmid part 7!); smaller and larger clones on all plates; further incubation over day since they were too small to be picked in the morning…

Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)

-          According to protocol from 10.7.13

-          16x MasterMix

-          Clones 1 – 10 picked, streaked out on LB^Amp-plate and used for inoculation of 25 µl reactions (blue tubes)

-          Controls: 1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and RBS-GFP-Terminator C12 (yellow tubes)

Concentration of dialysed protein samples

-       Put E1 samples from dialysis in vivaspin column

-       Spin column for 20min at 4000xg

-       Add 4ml of E2 to vivaspin and centrifuge for 20min at 4000xg

-       Resuspend and repeat centrifugation and resuspension

Determination of final protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H₂O) to 5µl of 1:20 diluted protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD₅₉₅ / Vx0,0536

SDS Page of dialysis and elution samples

-       Mix 15µl of samples  with 5µl Pab and 5µl buffer W

-       Incubation for 10min at 93°C

-       Mix each sample with 4µl protein marker

-       Load whole volume on 15% SDS-gel

-       Run for 5min at 90V and 1h at 120V

D1 | D2 | D3 | M | E1 | E2 |

Test run of plasmids purified on 12.7.13, vector and inserts on agarose gel

• part 7 plasmid, purified on 12.7.13
• pSB1C3 vector from part 7, purified on 11.7.13
• plasmid RBS-GFP-Terminator C12
• plasmid RBS-GFP-Terminator C12 test restriction with EcoRI
• inserts of DarR/riboswitches after 2nd digest with EcoRI
• 1% agarose-1xTAE-gel
• 3 µl 2 log ladder loaded on both sides of gel as a marker

Samples:

(for Riboswitch iGEM_41/42 and _42/43, there were too small amounts of PCR product left -> control was not possible…)

• Run at 100 V
• EtBr staining + destaining in water
• UV detection

Purification of DarR and Riboswitch inserts

• Qiagen PCR purification kit
• 500 µl PB used for each reaction
• 2x elution in pre-warmed HPLC water (25 µl)
• NanoDrop concentration measurements (for some samples, the concentration was measured twice, since first values for 1st and 2nd elution were a little strange… -> approximate average concentration can be used for further calculations)