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Team:TUDelft/3 August 2009
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===Sriram=== | ===Sriram=== | ||
| - | All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies | + | All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies from TSS buffer competent cells was less. However the concentration of DNA added plays a major role in the efficiency of transformation. The λp-R-GFP-T DNA isolated by miniprep had a concentration of 116.5 ng/µl which gave huge number of colonies compared to the diluted pSB1K3 with mRFP biobrick. The electro-transformation of λp-rep biobrick was successful and gave good colonies. |
Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies. | Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies. | ||
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===Calin=== | ===Calin=== | ||
Revision as of 07:18, 20 October 2009
Lab Notebook
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3 August 2009
Tim Weenink
I stole p(tetR) promotor and double terminator from the delay team to redo the !B and !C assemblies. The ligation products were run on Calin's gel (see below). The transformants were plated on Cam agar plates (unfortunatele only LB agar was left that seemed infected.) and stored in the 37 degrees C stove.
Sriram
All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies from TSS buffer competent cells was less. However the concentration of DNA added plays a major role in the efficiency of transformation. The λp-R-GFP-T DNA isolated by miniprep had a concentration of 116.5 ng/µl which gave huge number of colonies compared to the diluted pSB1K3 with mRFP biobrick. The electro-transformation of λp-rep biobrick was successful and gave good colonies.
Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies.
| Well No. | Sample Name | Expected Plasmid Size | Status |
| 1 | pTet-Upstream | ✔ | |
| 2 | RBS-cI-RBS-Downstream | ✔ | |
| 3 | pSB1C3-plasmid | ✔ | |
| 4 | RFP-Upstream | ✔ | |
| 5 | Term-Downstream | ✔ | |
| 6 | pSB1C3-plasmid | ✔ | |
| 7 | pLacI-Upstream | ✔ | |
| 8 | RBS-Downstream | ✔ | |
| 9 | pSB1C3 plasmid | ✔ | |
| 10 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 11 | TetR-Upstream | ✔ | |
| 12 | Term-Downstream | ✔ | |
| 13 | pSB1C3 plasmid | ✔ | |
| 14 | pTet-Upstream | ✔ | |
| 15 | Lock3C-Downstream | ✔ | |
| 16 | pSB1C3 plasmid | ✔ | |
| 17 | cI-Upstream | ✔ | |
| 18 | Term-Downstream | ✔ | |
| 19 | pSB1C3 plasmid | ✔ | |
| 20 | pLambdain-Upstream | ✔ | |
| 21 | RBS-GFT-T-Downstream | ✔ | |
| 22 | pSB1C3 plasmid | ✔ | |
| 23 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Calin
Tim pointed out that the plasmids should be linearized before doing the gel. Here are possible digestions on some of the parts:
| Backbone | Part | Unique Sites Present |
| pSB1A2 | oriT-R | AatII
AccI AflIII BglI BspLU11I BstAPI DrdI Eam1105I EcoRI FspI NspI PstI PvuI RsaI ScaI SgrAI SpeI SspI StyI TatI VspI XbaI XmnI ZraI |
| BBa_J61002 | strong promoter | AatII
AlwNI AvrII BglI BsiWI
BspLU11I BstXI DrdI EcoRI FspI HaeII HpaI NcoI NspI PflMI PshAI PstI PvuI PvuII RsrII ScaI SgrAI SpeI SspI TatI VspI XbaI XmnI ZraI |
| pSB1AK3 | ccdB | AatII
AccB1I AccI BamHI BanII BglI BspLU11I BsrGI BssHII BstXI BstZ17I BtrI ClaI DraIII DrdI Eam1105I EcoNI EcoRI FspI HaeII HincII HindIII MscI NruI NspI PflMI PstI ScaI SgfI SpeI SrfI XbaI XmnI ZraI |
| pSB4C5 | ccdB | AatII
AccI AclI AflII ApaLI BamHI BbeI BseRI BsrGI BssHII BstAPI BstXI BstZ17I BtrI Cfr10I EcoRI EcoRV HaeII HincII KasI NarI NdeI PstI PvuII SacI ScaI SfoI SmaI SpeI SphI SrfI StyI XbaI XmaI ZraI |
Growth on all plates. The pSB4C5 and R751 plates were parafilmed and placed in fridge.
Did linearization of oriT-R, promoter, pSB1AK3, and pSB4C5 using EcoRI.
Did digests of oriT-R (9uL) and pSB4C5 (4.5uL).
Did ligation CA and CB:
| Assembly name | position | Components | volume in µl |
| CA | Upstream | pTet-GFP | 2 |
| Downstream | oriT-R | 2 | |
| Backbone | pSB4C5 | 2 | |
| H20 | 11 | ||
| CB | Upstream | rbs-GFP-term | 2 |
| Downstream | oriT-R | 2 | |
| Backbone | pSB1C3 | 2 | |
| H20 | 11 |
Ran a 2% gel to check various parts and digests.
| Well | Part | Expected Plasmid Size | Status |
| 1 | oriT-R linear | 2079 | ✔ |
| 2 | oriT-R old digest | 2079 + ~278 | ✔ |
| 3 | oriT-R control | 2079 | ✔ |
| 4 | oriT-R new digest | 2079 + ~278 | ✔ |
| 5 | promo linear | 2983 | ✔ |
| 6 | promo control | 2983 | ✔ |
| 7 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 8 | pSB1AK3 linear | 3864 | ✔ |
| 9 | pSB1AK3 control | 3864 | ✔ |
| 10 | pSB4C5 linear | 3896 | ✔ |
| 11 | pSB4C5 control | 3896 | ✔ |
| 12 | pTet digest | 2079 + 937 | ✔ |
| 13 | GFP-gen digest | 2079 + 878 | ✔ |
| 14 | pSB1C3 digest | 2072 + 675 | ✔ |
| 15 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 16 | !B assembly ligation | circular | ? |
| 17 | !C assembly ligation | circular | ? |
Orr
Made some LB Agar medium and some agarose gel with pre-made TBE at concentration 1x.
