Team:Heidelberg/Notebook natural promoters
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* Test digest (EcoRI and PstI) of NFAT/NFkB promoter within the p31 plasmid (figure below)at 37°C for one hour. | * Test digest (EcoRI and PstI) of NFAT/NFkB promoter within the p31 plasmid (figure below)at 37°C for one hour. | ||
* NFAT#2 and NFAT#5 plasmids sent to sequencing. | * NFAT#2 and NFAT#5 plasmids sent to sequencing. | ||
- | [[image:HD09_31.08._NFkB_NFAT.gif|center|600px|thumb|'''Figure | + | [[image:HD09_31.08._NFkB_NFAT.gif|center|600px|thumb|'''Figure 2 1% agarose gel electrophoresis for a test digest of the NFkB/p31 plasmid and the NFAT/p31 plasmid.''' The NFkB/p31 and NFAT/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting NFAT insert should be 134 bp long and NFkB 179 bp. NFAT #2 and NFAT #5 seem to be successfully ligated plasmids, because there are small bands at ~ 150 bp. The lanes of NFkB #4 and NFkB #5 show a p31 plasmid band, which is 4919 bp long. But the NFkB inserts could not be recognized. Nevertheless, we will try to sequence them. All other lanes display definetly the wrong products.]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting NFAT insert should be 134 bp long and NFkB 179 bp. NFAT #2 and NFAT #5 seem to be successfully ligated plasmids, because there are small bands at ~ 150 bp. The lanes of NFkB #4 and NFkB #5 show a p31 plasmid band, which is 4919 bp long. But the NFkB inserts could not be recognized. Nevertheless, we will try to sequence them. All other lanes display definetly the wrong products.]] | ||
Revision as of 13:14, 19 October 2009
Natural PromotersContents
8-11-2009
8-18-2009
8-19-2009
8-20-2009
8-21-2009
8-24-2009
8-25-2009
8-26-2009
![]() Figure 1: 1% agarose gel electrophoresis of the PCR products of APOAIV and CYP1A1 promoter.The APOAIV and CYP1A1 promoter PCR products were analysed by agarose gel electrophoresis. Two reaction mixtures were used; one with Taq polymerase and the other one with Phu polymerase and with and without DMSO adding. Besides, the reaction mixtures of Phu have a varying concentration of MGCl2 (1-4 µl in a 50 µl reaction mixture). Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting CYP1A1 inserts should be 1229 bp long and APOAIV ~4200 bp. The "CYP1A1 Taq" and "CYP1A1 Taq + DMSO" are the only products, which have the right length. All the other lanes show an unspecific PCR. 8-27-2009
8-28-2009
8-29-2009
8-31-2009
![]() Figure 2 1% agarose gel electrophoresis for a test digest of the NFkB/p31 plasmid and the NFAT/p31 plasmid. The NFkB/p31 and NFAT/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting NFAT insert should be 134 bp long and NFkB 179 bp. NFAT #2 and NFAT #5 seem to be successfully ligated plasmids, because there are small bands at ~ 150 bp. The lanes of NFkB #4 and NFkB #5 show a p31 plasmid band, which is 4919 bp long. But the NFkB inserts could not be recognized. Nevertheless, we will try to sequence them. All other lanes display definetly the wrong products. 9-01-2009
9-02-2009
9-03-2009
9-04-2009
9-07-2009
9-08-2009
9-09-2009
![]() Figure ??: 1% agarose gel electrophoresis for a test digest of the CYP1A1/p31 plasmid. The CYP1A1/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (75 -20000 bp); Fermentas DNA 1kb Plus) is added into the gel for the validation of the DNA product length. The resulting CYP1A1 insert should be 1229 bp long. CYP1A1 #2, CYP1A1 #5 and CYP1A1 #8 seem to be successfully ligated plasmids, because there are small bands at ~ 1200 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other CYP1A1 inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp). 9-10-09
9-11-09
9-14-2009
9-15-2009
9-16-2009
9-17-2009
9-18-2009
9-20-2009
9-21-2009
9-22-2009
9-23-2009
9-24-2009
9-25-2009
9-26-2009
C-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #9, RARE #15, RARE #23, HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11, LDLR #12, LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 . ![]() Figure ??: 1% agarose gel electrophoresis of a test digest of the c-Jun/p31 plasmid constructs.The c-Jun/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting c-Jun insert should be 341 bp long. C-Jun #19, c-Jun #20, c-Jun #21 and c-Jun #22 seem to be successfully ligated plasmids, because there are small bands at ~ 300 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other c-Jun inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp). ![]() Figure ??: 1% agarose gel electrophoresis of a test digest of the RARE/p31 plasmid constructs.The RARE/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting RARE insert should be 212 bp long. The RARE inserts are difficult to recognize, because the JeT insert of the original plasmid is ~ 180 bp. Therefore, some good constructs are chosen: RARE #8, RARE #9, RARE #15 and RARE #23. ![]() Figure ??: 1% agarose gel electrophoresis of a test digest of the HMG CoA synthase/p31 plasmid constructs.The HMG CoA synthase/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HMG CoA synthase insert should be 363 bp long. All lanes show a p31 plasmid band, which is 4919 bp long. But the HMG CoA inserts could not be recognized. All lanes display definetly the wrong product (JeT, ~180 bp). ![]() Figure ??: 1% agarose gel electrophoresis of a test digest of the LDLR/p31 and HMG CoA synthase/p31 plasmid constructs.The LDLR/p31 and HMG CoA synthase/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11 and LDLR #12 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp). ![]() Figure ??: 1% agarose gel electrophoresis of a test digest of the LDLR/p31 and HMG CoA synthase/p31 plasmid constructs.The NFkB/p31 and NFAT/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp). 9-28-2009
![]() Figure ??: 1% agarose gel electrophoresis for a test digest of the HSP70/p31 plasmid construct.The HSP70/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HSP70 insert should be 402 bp long. HSP70 #9 seem to be a successfully ligated plasmid, because there is a small bands at ~ 400 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HSP70 inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp). 9-29-2009
Notebook information about the measurement of natural promoters is found in the Notebook Measurement and Cell Culture???. LINK 10-12-2009
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