Team:Munich/engineeringrecbcd.html
PCR amplify RecBCD WT
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR, Gel extraction, Restriction digest, PCR purification, Ligation |
| Notes: | RecC: RecC_3'UTR_SapI_rv, RecC_BB-P_XbaI_fw, AT: 71°, ET: 70s RecBD: RecB_SapI_fw, RecD_BB-S_SpeI_rv, AT: 72°, ET: 108 s Digests: RecC: SapI & XbaI; RecBD: SapI SpeI Restriction digest with RecC(WT): SapI & XbaI; RecBD(WT): SapI SpeI |
| Results: | see below |
PCR amplify RecBCD His-tag
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR, Restriction digest, PCR purification |
| Notes: | RecC: RecC_C-His_5'UTR_BBP_XbaI_fw
RecB_5'UTR_SapI_rv
AT: 72°
ET: 70s
RecBD: RecB_N-His_SapI_fw RecD_C-His_BBS_SpeI_rv AT: 69° ET: 108 s Digests: RecBD SapI XbaI, RecC-his: SapI XbaI |
| Results: | see below |
PCR amplify RecBCD 5' homology regions
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR |
| Notes: | RecBCD_KI_5HA_extract_fw RecBCD_KI_5HA_extract_rv AT: 62° ET: 15 s |
| Results: | see below |
PCR amplify RecBCD 3' homology regions
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR |
| Notes: | RecBCD_KI_3HA_extract_rv RecBCD_KI_3HA_extract_fw AT: 62° ET: 15 s |
| Results: | see below |
PCR amplify resistance cassette for RecBCD knockout
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR, |
| Notes: | CAT_RecBCD-KO_5HA_short_fw CAT_RecBCD-KO_5HA_short_rv AT: 51° ET: 20s |
| Results: | see below |
PCR amplify resistance cassette for RecBCD knockout (2)
2018/06/11| Participants: | Enikö Baligács, Rita Olenchuk, Julia Mayer |
| Protocol: | PCR, |
| Notes: | CAT_RecBCD-KO_5HA_long_fw CAT_RecBCD-KO_5HA_long_rv AT: 55° ET: 20s |
| Results: | see below |
Redo: PCR amplify RecBD His-tag
2018/06/11| Participants: | Enikö Baligács |
| Protocol: | PCR, Agarose Gel, Gelextraction |
| Notes: |
RecBD: RecB_N-His_SapI_fw
RecD_C-His_BBS_SpeI_rv
AT: 69°
ET: 108 s
|
| Results: | 51,4ng/ul |
Assemble pSB1C3_RecBCD-his
2018/06/11| Participants: | Enikö Baligács |
| Protocol: | Restriction digest, PCR purification, Ligation |
| Notes: | digest with SapI SpeI |
Prepare pSB1C3 backbone
2018/06/11| Participants: | Dominic Schwarz |
| Protocol: | Electrocompetent transformation, Mini Prep, Restriction digest, Agarose gel |
| Notes: | template: pSB1C3-J04450 (iGEM BioBrick Kit plate 7 Well 23 O) |
| Results: | agarose gel wasnt prepared properly leading to loss of samples. We decided to redo the experiment |
Amplify Lac-Operon parts from pSB1C3 backbone
2018/06/11| Participants: | Dominic Schwarz |
| Protocol: | PCR, Agarose gel, Restriction digest |
| Notes: | template: pSB1C3-J04450 (iGEM BioBrick Kit plate 7 Well 23 O) Digests with: XbaI, SpeI and EcoRI, PstI for future assemblies Lac Promoter: BBP_EcoRi_fw & LacO_XbaI_rv (57°C, 1min) Lac terminator: Term_SpeI_fw & BBS_PstI_rv (65°C, 1min) |
| Results: | see below |
Assemble pSB1C3_lacO_RecBCD_lacT
2018/06/14| Participants: | Dominic Schwarz |
| Protocol: | Ligation, Chemical transformation, Mini Prep, Sequencing |
| Notes: | template: pSB1C3-J04450 (iGEM BioBrick Kit plate 7 Well 23 O) Digests with: XbaI, SpeI and EcoRI, PstI for future assemblies |
| Results: | colonies but sequencing gave no results. We decided to test the PCR primer on a gel. Resequencing with VF2 and VR and blast analysis showed contamination with pBR322. After reevaluating the design of the plasmid with Thomas (supervisor) we realized there is an error. We redesigned the plasmid and plan to assemble it with gibson assembly. |
Testing RecBCD Primers
2018/06/22| Participants: | Katja Neishsalo |
| Protocol: | Agarose gel, |
| Results: | As expected all primers are ok :) PIC |
Midi prep pSB1C3_mRFP
2018/07/31| Participants: | Sophie von Schönberg, Ariane Krus, Franziska Winzig |
| Protocol: | Midi Prep, Restriction digest, Agarose gel |
| Notes: | we needed lots of backbone and linear DNA for other experiments, which is why we did this DNA preparation. obtained samples were controlled by restriction digest with KpnI, PstI : 500bp, 2672bp |