Team:Freiburg bioware/Notebook/July

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*SDS-Gel: [[Image:Freiburg09_010709_Exprssion2_trim.jpg|none|thumb|SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Tt 1h..2h..4h, T7+Aa 1h..2h..4h, BL21+Aa 1h..2h..4h|400x400px]]  

*SDS-Gel: [[Image:Freiburg09_010709_Exprssion2_trim.jpg|none|thumb|SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Tt 1h..2h..4h, T7+Aa 1h..2h..4h, BL21+Aa 1h..2h..4h|400x400px]]  

*PCR with the purified PCR product from the 30.06.09 as template. Different program: nr. 25 IGEM.AM -> no result. next step should be to do a gradient PCR this afternoon. Just talk to Sven to get the details.

*PCR with the purified PCR product from the 30.06.09 as template. Different program: nr. 25 IGEM.AM -> no result. next step should be to do a gradient PCR this afternoon. Just talk to Sven to get the details.

*idea from Sven: do a westernblot on the isolated proteins from the AGO-Expression to find out whether the AGO-protein is present or not.

*idea from Sven: do a westernblot on the isolated proteins from the AGO-Expression to find out whether the AGO-protein is present or not.

*gradient PCR from 60°-70°C with 8 tubes each of vector 258 diluted and undiluted, we put it in the cold room -> gel has to be done tomorrow

*gradient PCR from 60°-70°C with 8 tubes each of vector 258 diluted and undiluted, we put it in the cold room -> gel has to be done tomorrow

*we made 100ml 1M Tris-HCl pH=6,8

*we made 100ml 1M Tris-HCl pH=6,8

-alterations of PCR-program: only one step but 30 times, with temperature gradient 55-65°C (Sven can help)

-alterations of PCR-program: only one step but 30 times, with temperature gradient 55-65°C (Sven can help)

*AGO: expression of Aa and Tt (T7+Tt, T7+Aa, BL21+Aa) at 27°C. samples after 0, 1.5 and 4 hours

*AGO: expression of Aa and Tt (T7+Tt, T7+Aa, BL21+Aa) at 27°C. samples after 0, 1.5 and 4 hours

*plasmid preparation of inoculated labvector 258

*plasmid preparation of inoculated labvector 258

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*PCR of the CAT-Vektor (258) with new dNTP-Mix and a anealing-gradient from 50°C to 65°C -> bands at every temperature with expected size

*PCR of the CAT-Vektor (258) with new dNTP-Mix and a anealing-gradient from 50°C to 65°C -> bands at every temperature with expected size

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[[Image:Freiburg09_030709_Exprssion3001.jpg|none|thumb|SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Aa 1h..2h..4h, T7+Tt 1h..2h..4h, BL21+Aa 1h..2h..4h|400x400px]]

[[Image:Freiburg09_030709_Exprssion3001.jpg|none|thumb|SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Aa 1h..2h..4h, T7+Tt 1h..2h..4h, BL21+Aa 1h..2h..4h|400x400px]]

*we did a agarose gel from the pcr product, cut the band and eluted the DNA. As the concentration only was about 50 ng/µl we did a new gel with 3 times the amount on pcr product (150 µl). We cut the bands, eluted the DNA and got a concentration from about 100 ng/µl. We used both eluted DNA samples for the restriction digest with HindIII and MfeI (4 hours at 37°C).

*we did a agarose gel from the pcr product, cut the band and eluted the DNA. As the concentration only was about 50 ng/µl we did a new gel with 3 times the amount on pcr product (150 µl). We cut the bands, eluted the DNA and got a concentration from about 100 ng/µl. We used both eluted DNA samples for the restriction digest with HindIII and MfeI (4 hours at 37°C).

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*digestion of the pMal-Vektor with HindIII and MfeI (both from the lab freezer), didn't work. Maybe the MfeI enzyme was bad (expired 2005)

*digestion of the pMal-Vektor with HindIII and MfeI (both from the lab freezer), didn't work. Maybe the MfeI enzyme was bad (expired 2005)

*started expression of AGO at 20°C. Took samples at 0, 2, 4 and 8 hours centrifuged them, decanted the supernatant and stored the pellets at -20°C in the box for Sascha.

*started expression of AGO at 20°C. Took samples at 0, 2, 4 and 8 hours centrifuged them, decanted the supernatant and stored the pellets at -20°C in the box for Sascha.

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*new digest of pMAL expression vector and CAT gene with MfeI and HindIII for four hours

*new digest of pMAL expression vector and CAT gene with MfeI and HindIII for four hours

*agarose gel with these two samples -> bands of CAT gene (probably cut) around 700 bp and pMAL vector around 5kb and MalE gene around 1,4kb could be seen, bands of CAT and vector were cut out

*agarose gel with these two samples -> bands of CAT gene (probably cut) around 700 bp and pMAL vector around 5kb and MalE gene around 1,4kb could be seen, bands of CAT and vector were cut out

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*Western: [[Image:Freiburg09 20090709 1343 anti-His.JPG|none|thumb|Westernblot of the AGOexpression; Lanes: empty lane, Tt in BL21 DE3 at 8h and 0h, Aa in BL21 DE3 at 8h and 0h, Tt in T7 at 8h and 0h, Aa in T7 at 8h and 0h 500µl culture each|400x400px]]

*Western: [[Image:Freiburg09 20090709 1343 anti-His.JPG|none|thumb|Westernblot of the AGOexpression; Lanes: empty lane, Tt in BL21 DE3 at 8h and 0h, Aa in BL21 DE3 at 8h and 0h, Tt in T7 at 8h and 0h, Aa in T7 at 8h and 0h 500µl culture each|400x400px]]

*SDS page, Coomassie staining [[Image:Freiburg09_090709_SDSpage018.jpg|none|thumb|SDS page of the AGOexpression; Lanes: Protein marker NEB, Aa in BL21 de3 waste fraction, fraction 1, fraction 2, fraction 3, Aa in T7 waste fraction, fraction 1, fraction 2, fraction 3, fraction 4|400x400px]]

*SDS page, Coomassie staining [[Image:Freiburg09_090709_SDSpage018.jpg|none|thumb|SDS page of the AGOexpression; Lanes: Protein marker NEB, Aa in BL21 de3 waste fraction, fraction 1, fraction 2, fraction 3, Aa in T7 waste fraction, fraction 1, fraction 2, fraction 3, fraction 4|400x400px]]

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*Trafo of the new Ligation showed only few colonies and the old ligation showed no colonies on CM or CM + IPTG -> inoculation and sequencing of clones on positive plates on monday   

*Trafo of the new Ligation showed only few colonies and the old ligation showed no colonies on CM or CM + IPTG -> inoculation and sequencing of clones on positive plates on monday   

*Trafo of His-Tag, Strep-Tag, Split-Linker showed good results; GGGs-Linker showed no grown colonies -> inoculation on monday

*Trafo of His-Tag, Strep-Tag, Split-Linker showed good results; GGGs-Linker showed no grown colonies -> inoculation on monday

*inoculation of His, Strep, Split-Linker in pMa (Amp)

*inoculation of His, Strep, Split-Linker in pMa (Amp)

*inoculation of the ligation-trafo from last week (CAT)

*inoculation of the ligation-trafo from last week (CAT)

*we prepared 1l of 2YT-medium

*we prepared 1l of 2YT-medium

*Plasmid-Preparation of His-Tag, Strep-Tag, Split-Linker, Ligation 9.7. (neu2), Ligation 9.7. (alt), Ligation 8.7. (100 µl and rest)

*Plasmid-Preparation of His-Tag, Strep-Tag, Split-Linker, Ligation 9.7. (neu2), Ligation 9.7. (alt), Ligation 8.7. (100 µl and rest)

*Digestion of FOKI, FOKA with XbaI and PSTI and SPLIT- Linker with SpeI, PstI.

*Digestion of FOKI, FOKA with XbaI and PSTI and SPLIT- Linker with SpeI, PstI.

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*centrifuged the induced Tt/AGO cells at 4°C, 15 min, 4000rpm, discarded the supernatant and put it in the -20°C freezer  

*centrifuged the induced Tt/AGO cells at 4°C, 15 min, 4000rpm, discarded the supernatant and put it in the -20°C freezer  

*new digest of Split, FOKa and FOKi -> used wrong tubes (old digest) -> sufficient labelling on the tubes (part, what was done with it, date) necessary!!!  

*new digest of Split, FOKa and FOKi -> used wrong tubes (old digest) -> sufficient labelling on the tubes (part, what was done with it, date) necessary!!!  

-purification of AGO with His/Ni column

-purification of AGO with His/Ni column

*digestion of Split, FOKa and FOKi + gelextraction

*digestion of Split, FOKa and FOKi + gelextraction

*sequenzing of Lig 8.7. "neu, alt, 100"

*sequenzing of Lig 8.7. "neu, alt, 100"

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* 5 l LBmedia prepared

* 5 l LBmedia prepared

* we got a look at the ssDNA of the M13 phage, there is no Fok restriction site.  

* we got a look at the ssDNA of the M13 phage, there is no Fok restriction site.  

*SDS-Gel purification and commassie staining of the purified Aa and Tt AGOs (Samples W2-4, E1-6)

*SDS-Gel purification and commassie staining of the purified Aa and Tt AGOs (Samples W2-4, E1-6)

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* made TBS buffer (100ml)

* made TBS buffer (100ml)

* start preparation of M13 phage ssDNA :overnight breeding of ER2738

* start preparation of M13 phage ssDNA :overnight breeding of ER2738

* made a glycerol stock of ER2738 Tet

* made a glycerol stock of ER2738 Tet

*transformation from yesterday (20.07.) plated on lb-amp again: 100 µl and centrifuged residue

*transformation from yesterday (20.07.) plated on lb-amp again: 100 µl and centrifuged residue

*start of documenting the cloning procedures like the design of primers and expressionvector (cloning strategies of genIII and the order of the ligation of the Fok, linker etc has to follow) in the new folder: cloning strategies

*start of documenting the cloning procedures like the design of primers and expressionvector (cloning strategies of genIII and the order of the ligation of the Fok, linker etc has to follow) in the new folder: cloning strategies

* precipitation of phage M13 particles

* precipitation of phage M13 particles

*plasmidpreparation of Ligation (pMAL&CAT) neu 1 & 2, Split linker, Digoxigenin 1 & 2, Lipocalin 1 & 2, Fok/Split Linker 1 & 2

*plasmidpreparation of Ligation (pMAL&CAT) neu 1 & 2, Split linker, Digoxigenin 1 & 2, Lipocalin 1 & 2, Fok/Split Linker 1 & 2

*Glycerolstocks of Ligation neu 1 & 2 (Sven did stocks of Foka & Foki, don't know where he put it)

*Glycerolstocks of Ligation neu 1 & 2 (Sven did stocks of Foka & Foki, don't know where he put it)

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*preparation of Buffers for anion exchanger

*preparation of Buffers for anion exchanger

*picked colonies of the plates: Trafo FokA(21.07.09) and Ligation FokA-Split(20.07.09) and transfered into 3ml LB-Amp

*picked colonies of the plates: Trafo FokA(21.07.09) and Ligation FokA-Split(20.07.09) and transfered into 3ml LB-Amp

stored in refrigerator(Premium)

stored in refrigerator(Premium)

* complete precipitation of phage M13 particles

* complete precipitation of phage M13 particles

* phage stock in -80°C fridge

* phage stock in -80°C fridge

* ssDNA in -20°C fridge

* ssDNA in -20°C fridge

*design and order of Gen-III Primers

*design and order of Gen-III Primers

*design and order of Short-, Middle- and Long-Linkers for Fok

*design and order of Short-, Middle- and Long-Linkers for Fok

*transformation of ligation from 22.07.09:<br>

*transformation of ligation from 22.07.09:<br>

- 50µl XBlue + 5µl ligation His-Lipo<br>

- 50µl XBlue + 5µl ligation His-Lipo<br>

--> 37°C o/n

--> 37°C o/n

* counted colonies from transformation (23.07.09):

* counted colonies from transformation (23.07.09):

- BL 21 de3 + pUC: 0 colonies<br>

- BL 21 de3 + pUC: 0 colonies<br>

*Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo

*Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo

*preparation of 100 ml LB+Chloramphenicol and 100ml LB+Tetracyclin

*preparation of 100 ml LB+Chloramphenicol and 100ml LB+Tetracyclin

*order of 2 oligos for the M13mp18 ss DNA (there are 4 Fok restriction sites) to cut at two sites resulting in fragments of about 890 and 6360 bp with Fok

*order of 2 oligos for the M13mp18 ss DNA (there are 4 Fok restriction sites) to cut at two sites resulting in fragments of about 890 and 6360 bp with Fok

*Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split in 5 ml LB+antibiotics (this time on the shaker)

*Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split in 5 ml LB+antibiotics (this time on the shaker)

*preparation of 75 ml LB+Ampicilin

*preparation of 75 ml LB+Ampicilin

*The Primers for Gene III have arrived

*The Primers for Gene III have arrived

*Plasmidpreparation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split -> very low concentrations except for His-Lipo and Strep-Dig (just low) -> made a digest with both and pMal-CAT (9.7. clon 2.1)

*Plasmidpreparation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split -> very low concentrations except for His-Lipo and Strep-Dig (just low) -> made a digest with both and pMal-CAT (9.7. clon 2.1)

*new inoculations of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split

*new inoculations of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split

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* plasmidprep Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split

* plasmidprep Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split

* glycerol stock of Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split

* glycerol stock of Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split

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'''Plan for today'''

'''Plan for today'''

*Nucleic acid

*Nucleic acid

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