2010-07-14
Julia and Katarina
Ran PCR 1-5.
1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.
Expected fragment length: 748 bp.
2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus.
Expected fragment length: 762 bp.
3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus.
Expected fragment length: 755 bp.
4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus.
Expected fragment length: 769 bp.
5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.
Expected fragment length: 757 bp.
K2. Control K2.
Julia, Katarina and Malin
Verified PCR 1-5: measured concentration by nanodrop and checked if the fragments were the correct length by running on a 2 % agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low Range).
PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths. Number 4 however did not work. Only a fragment around 80 bp was visible, probably a primer dimer.
Concentrations:
1. 443.9 ng/µl
2. 371.0 ng/µl
3. 361.7 ng/µl
4. --
5. 377.6 ng/µl
A. 451.1 ng/µl (snf1)
B. 438.7 ng/µl (snf4)
C. 408.9 ng/µl (SAMS)
1-5 and A-C were diluted to 1 ng/µl.
Ran PCR of I, II, III, IV and 4.
4.2. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment length: 769 bp. (second try)
I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.
II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.
III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected fragment length: 1708 bp. Final Product!
IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.
K3. Control
Karl and Malin
Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)
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2010-07-13
Julia and Lokesh
Ran the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration (1ng/µl) for the next PCR reactions.
Verified the PCR products and the digested miniprepped EYFP and ECFP by running an agarose gel which showed good results. Expected fragment lengths were received. The SAMS-peptide however was too short to be visible on the gel (67 bp).
Prepared for PCR 1-5. The miniprepped ECFP and EYFP were diluted to a stock concentration of 1 ng/ ml to use for the PCR. The concentrations of the first PCR product were measured using nanodrop and then diluted to 1 ng/ml.
snf1: 20.5 ng/ml
snf4: 122.6 ng/ml
SAMS: 114.9 ng/ml
Karl and Adnan
We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.
2010-07-12
Katarina and Julia
Amplification of genomic SNF1 DNA by PCR. All primers were resuspended in mq-water according to the volume on the lable, the final concentration 100 pg/ml. The primers were then diluted 10x and the SAMS-peptide templates were diluted 1000x. PCR reactions were run amplifying subunits Snf1 and Snf4 and also the SAMS-peptide. YPD medium and agar plates were prepared (yeast).
Adnan and Kemal
Miniprep purification of EYFP and ECFP plasmides from overnight culture. The concentrations were measured using Nanodrop:
ECFP1: 99 ng/ml
ECFP2: 89.9
EYFP1: 104, 7
EYFP2: 172,5
The concentrations were verified with electrophoresis. The plasmids were cut with restriction enzymes EcoRI and Pst1.
ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.
EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.
2010-07-11
Katarina
Made overnight cultures with colony 3 and 4 plasmid (not single cultures) and twp each of EYFP and ECFP.
2010-07-10
Julia
Moved plated bacteria from incubator to refrigerator.
2010-07-09
Malin and Julia
The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.
Adnan and Katarina
Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.
2010-07-08
Malin, Julia and Peidi
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.
2010-07-07
Adnan, Katarina and Julia
As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 & 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel were very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.
2010-07-06
Malin, Adnan and Peidi
Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.
2010-07-05
Malin and Katarina
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1. |
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