Team:Heidelberg/Notebook natural promoters

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Revision as of 11:25, 15 October 2009

Notebook

8-11-2009

Plasmids and primers with the specicfic natural promoters had been ordered

8-18-2009

Start with natural promoter project
The two promoters, Apolipoprotein A-IV (APOAIV) and Cytochrome P450 1A1 (CYP1A1) promoter, were amplified by PCR using human genomic DNA.

8-19-2009

APOAIV promoter PCR product was digested with NheI and SpeI
The backbone plasmid (p31; ~5000 bp) was also digested with the same restriction enzymes and it was subsequent digested with SAP

8-20-2009

CYP1A1 promoter has the internal restriction site, NheI,...waiting for another CYP1A1 reverse primer with the restriction site PstI instead of NheI.
The plasmid (p31) and the insert (APOAIV promotor) were purified and ligated.
The ligated product was transformed into DH5alpha cells

8-21-2009

Picked eight colonies of p31/APOAIV plates and did a mini-preparation (miniprep)
Test digest of the eight miniprep's using the restriction enzymes SpeI and NheI

IMAGE of digest

There is an appropriate band fpr the APOAIV promoter (~4000bp length).
Miniprep of colony p31/ApoAIV #1, p31/ApoAIV #2 and p31/ApoAIV #3

8-24-2009

The plasmids p31/APOAIV #3 and p31/APOAIV #1 will be sequenced by GATC

8-25-2009

APOAIV sequence did not match the published sequence
The test digest did not yield expected fragments--> we need to troubleshoot and repeat PCR from genomic DNA

8-26-2009

Troubleshooting PCR for CYP1A1 and APOAIV using Taq mastermix and Phu with freshly mixed ingredients. We also used various MgCl2 concentrations (1, 2, 3, 4 ul of 25 mM MgCl2 per 50ul reaction) and performed the reaction with and without the addition of DMSO.
Analytic gel below shows CYP1A1 with Taq -/+ DMSO in the first two lanes with bands at the expected height of around 1200 bp. Lanes 1-10 are different CYP1A1 reactions, lanes 11-20 are APOAIV reactions. APOAIV shows only nonspecific bands, has to be further optimized using gradient PCR or new primers.

8-27-2009

Test digest of CYP1A1 insert (1.2 kb) with NheI (digest at position 614 bp)
PCR of NFkB and NFAT plasmids to amplify promoters from Oakes plasmids p46 and p47. Amplified using PCR high annealing protocol:

Step	Time	Temperature
1.	4 min	95°C
2.	30s	95°C
3.	45s	59°C
4.	2 min	72°C
5.	Go to step 2	24 times
6.	30s	95°C
7.	45s	66°C
8.	2 min	72°C
9.	for ever	4°C

8-28-2009

Digest of CYP1A1 insert with PstI and SpeI and NFkB and NFAT inserts with PstI and EcoRI
Digest of plasmid p31 with PstI/SpeI and PstI/EcoRI with SAP digest afterward
PCR purification of digested inserts, gel purification of plasmid digests

8-29-2009

Ligation of p31 and promoters (NFAT, NFkB)
Transformation of ligated plasmids (p31) with the promoters NFAT and NFkB.

8-31-2009

Miniprep of p31 plasmid + NFAT/NFkB promoter
Ligation of CYP1A1 promoter and p31 plasmid
Test digest of NFAT/NFkB promoter within the p31 plasmid (figure below)
NFAT and NFkB promoters sent to sequencing

Figure ??: 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) 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

Transformation of CYP1A1/p31 plasmid

9-02-2009

Minipreparation of CYP1A1 colonies (six colonies)
Test digest of the ligated CYP1A1 construct -> no success
APOAIV promoter: PCR of genomic DNA with old conditions

9-03-2009

Analysis with agarose gelelectrophoresis of the APOAIV-PCR product -> no success
Digest of CYP1A1 promoter and plasmid p31 with the restriction enzymes, SpeI and PstI
PCR Purification of the CYP1A1 promoter and gel extraction of p31.
Ligation of digested CYP1A1 promoter and p31 (over night) at 16°C.

9-04-2009

PCR with the plasmid p52 to isolate the HSP70 promoter
PCR with the plasmid p46 to isolate the promoter with NFkB binding sites
Digest of the plasmid p31 with the restriction enzymes, EcoRI and NheI

9-07-2009

Digest of promoter with NFkB binding sites (NFkB_prom) with NheI and EcoRI
Digest of half of the volume of the HSP70 promoter with EcoRI and NheI
Digest of half of the volume of the HSP70 promoter with PstI and SpeI
Ligation of the digested NFkB_prom and the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09)
Ligation of the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09)
Ligation of the HSP70 promoter (SpeI, PstI) with p31 (SpeI, PstI; 09/03/09)
Transformation of the ligated products (above) and the ligated product of CYP1A1 (09/03/09)

9-08-2009

Pick some colonies of transformed bacteria
Miniprep
Test digest

9-09-2009

Gel electrophoresis image of the test digest:

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CYP1A1_promoter/p31 construct was included in three positive colonies. Therefore this construct is ready for mutagenesis PCR to remove the NheI restriction site at 614 bp
The others have not turned out satisfactory.

10-09-09

The sequence of CYP1A1 #2 and #5 are correct.
Plate out the addgene plasmids: pJC6-GL3, SBE4-Luc, pGL3-RARE-Luc, pGL3-NFAT-Luc, PUMA-Frag1-Luc, pLDLR-Luc, pHMGCS-Luc

11-09-09

Pick some colonies of the plates (10.09.09) and miniprep.

14-09-2009

PCR of HSP70, NFAT and NFkB
Digest of the PCR products by NheI and EcoRI
Digest of p31_BBB by NheI and EcoRI
Send to sequencing: pJC6-GL3, SBE4-Luc, pGL3-RARE-Luc, pGL3-NFAT-Luc, PUMA-Frag1-Luc, pLDLR-Luc, pHMGCS-Luc
Mutagenesis PCR of CYP1A1/p31 #5

15-09-2009

PCR-Purification with HSP70, NFkB and NFAT
Gel-extraction of p31_BBB, which was digested by NheI and EcoRI
Ligation: Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB
PCR troubleshooting with NFAT and NFkB
Amplification of the p52 plasmid
CYP1A1-muta/p31 PCR product was digested by DpnI
Transformation of the three ligations (below: Hsp70/p31, NFAT/p31 and NFkB/p31) and CYP1A1-muta/p31

16-09-2009

Arrival of the sequencing results of the ordered Addgene plasmids
Design of primers for c-Jun promoter, LDL receptor promoter, Retinoic Acid Receptor Response Element
Unfortunaltely, PUMA promoter is not used because of a PstI restrcition site and it is no time for mutagenesis PCR
p52 (with HSP70 insert) is amplified in bacteria
Miniprep of the following ligation : Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB
And test digest of the ligations with EcoRI and PstI and there is something on the agarose gel
The PCR troubleshooting with NFAT and NFkB were analysed by a agarose gel and there are not the right bands

BILD???

17-09-2009

Miniprep of p52 (with HSP70 insert) and send to sequencing
Ligation of NFAT and NFkB (of troubleshooting PCR) with p31 plasmid
No colonies of CYP1A1-muta -> try again the mutagenesis PCR (with special mutagenesis Kit)

18-09-2009

Design of primers for the HSP70 insert by analysis of p52 sequence
Second mutagenesis PCR of CYP1A1
Transformation of NFAT and NFkB ligations

20-09-2009

pick colonies of NFAT and NFkB

21-09-2009

PCR of RARE, LDLR, HMG CoA synthase, c-Jun
Miniprep of NFAT and NFkB and test digest

22-09-2009

Tranformation of CYP1A1-muta
Analysis of the the following PCR products by agarose gel electrophoresis: RARE, LDLR, HMG CoA synthase, c-Jun.

BILD!!!

Diegst of these PCR products by the restriction enzymes, NheI and EcoRI.

23-09-2009

Gel extraction of RARE, LDLR, HMG CoA synthase and c-Jun insert.
Ligation of these extracted inserts with the digested (NheI and EcoRI) plasmid p31.
PCR of the p52 (with HSP70 promoter insert) and analysis of the PCR product by agarose gel electrophoresis.
Digest of the HSP70 promoter by NheI and EcoRI.
Transformation of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct.

24-09-2009

Ligation of the HSP70 promoter with the digested (NheI and EcoRI) p31 plasmid.
Transformation of the HSP70 promoter/p31 construct.
Colonies of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct are picked.

25-09-2009

Miniprep of RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct.
Colonies of the HSP70 promoter/p31 construct are picked.

26-09-2009

Miniprep of the HSP70 promoter/p31 construct.
Test digest (NheI and EcoRI) of RARE, c-Jun, LDLR and HMG CoA synthase

28-09-2009

Test diegst (NheI and EcoRI) of HSP70

@@ Line 118: / Line 118: @@
 * Test digest of NFAT/NFkB promoter within the p31 plasmid (figure below)
 * NFAT and NFkB promoters sent to sequencing
-[[image:HD09_31.08._NFkB_NFAT.gif|center|720px|thumb|'''Figure ??: 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 -
+[[image:HD09_31.08._NFkB_NFAT.gif|center|600px|thumb|'''Figure ??: 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 -
 bp) 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.]]