Team:UNAM Genomics Mexico/Notebook/ANDSugar
From 2012.igem.org
Arabinose/Xylose AND Gate
Nanotubes!! | The logic | Random info |
JUNE
06/07/12
PY BROTH 10g salt/L
PSB2K3 kanamicin
We transformed RFP E1010.
plate 1 18F
plate 2 17E
Stock 50 mg/mL
E.coli 1/1000
TRANSFORMATION PROTOCOL.
We made liquid cultures with colonies LIQUID CULTURE.
We extracted plasmids PLASMID EXTRACTION PROTOCOL.
We ran a gel to check the extraction GEL ELECTROPHORESIS PROTOCOL.
06/12/12
We made glycerols of the bacteria
GLYCEROL PROTOCOL .
We transformed plasmid pHp45 TRANSFORMATION PROTOCOL.
06/13/12
We ran gel and extracted from gel LIQUID CULTURE.
We made liquid cultures LIQUID CULTURE and lysed [LYSIS PROTOCOL].
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06/14/12
We extracted from gel LIQUID CULTURE.
We made glycerols of pHp45 Ω GLYCEROL PROTOCOL .
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450 PSB4A5 Am (ampicillin) 1I BBa_J04450 AraC BBa_C0080
2012 14L plate 1 pSB2K3 Km+
2011 14L plate 1 pSB2K3 Km+
2012 14L plate 1 pSB2K3 Km+
TRANSFORMATION PROTOCOL.
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06/15/12
> Digested pHp45 Ω with E/P LIQUID CULTURE.
> We extracted plasmid with kit.
> Ran a gel GEL ELECTROPHORESIS PROTOCOL . (5)
>Extracted from gel LIQUID CULTURE.
ARAC
>From the overnight plate we prepared liquid culture LIQUID CULTURE.
>We left them incubating overnight.
>We left a plate (LB Km DH5α C0080 and a control).
06/18/12
>Plasmid extraction AraC with kit.
>C0080-psB2K3 915 bp
>Streaked 4 LB Km 30 plates DH5α psB2K3.
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red.
>Ran gel with Spr/Strr (eppendorf -20ºC) GEL ELECTROPHORESIS PROTOCOL.
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04.
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 GLYCEROL PROTOCOL.
>Digested C0080 X,S.
>Digested B0014 E,P LIQUID CULTURE.
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06/19/12
>Digested B0014 with E, P and with E,X. LIQUID CULTURE.
>Ran gels with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL .
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation.
C0080 is in the first lane.
>Extracted from gel C0080 X,S LIQUID CULTURE.
>Made glycerols from 4 plates LB Km DH5α pSB2K3 GLYCEROL PROTOCOL .
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS].
AmyE 5’ 18K plate 3 2010, 2011, 2012
AmyE 3’ 18M plate 3 2010, 2011, 2012
>Digested B0014 with E,X and E,P again LIQUID CULTURE.
06/22/12
>Ran gels with digestions B0014 with E,X and E,P GEL ELECTROPHORESIS PROTOCOL.
>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL].
06/25/12
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS].
06/26/12
>Ran gel with psB2K3 and psB4A5 GEL ELECTROPHORESIS PROTOCOL .
06/27/12
>Transformed with plasmid B0079 1576 bp psB1A2 12A TRANSFORMATION PROTOCOL.
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011
AmyE 5’ grew 2 colonies.
06/29/12
>We did a DH5α K143001 Km30 Amp 100 glycerol GLYCEROL PROTOCOL 07.
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control
These were both plated in 2 plates each.
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made.
>Liquid cultures LIQUID CULTURE.
2 tubes DH5α K143001 Km30 Amp 100
2 tubes DH5α K143002 Km30 Amp 100
2 tubes DH5α B0079 Amp 100
1 tube LB Km 30 Amp 100 control
1 tube LB Amp 100 control
>From the 6 tubes we extracted plasmid from kit.
JULY
07/02/12
>Digestions LIQUID CULTURE.
B0079 digestion with S,P
K143001 with S,P
K143002 with S,P
>PCR’s
•AraC
•Cassete ΩSpr/Strr
PCR PROTOCOL
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-
07/03/12
>After Cassete ΩSpr/Strr PCR we ran a gel GEL ELECTROPHORESIS PROTOCOL . (8)
>Ran gel with digestions from yesterday. (9)
>Did band extractions LIQUID CULTURE.
>Stored at -20ºC.
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix [PCR PROTOCOL].
>Left digesting with E,S LIQUID CULTURE.
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07/04/12
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL].
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL].
>Transformed ligation and left overnight plated TRANSFORMATION PROTOCOL.
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
>Digested with PstI LIQUID CULTURE.
07/06/12
4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown.
Ran a gel with yesterday’s digestions to chek if they were done properly (10)
GEL ELECTROPHORESIS PROTOCOL .
Ran another gel with the rest of the samples.
Extracted GusA fragment
LIQUID CULTURE.
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated LIGATION PROTOCOL.
Repeated ΩSpr/Strr PCR.
07/07/12
Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 .
07/08/12
Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002.
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-
07/09/12
From yesterday’s transformations only one colony grew.
From the previous transformation only 2 colonies grew.
These 3 were streaked in 3 plates:
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km 30 Sp 100 control.
Did liquid cultures in 3 tubes:
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km30 Sp 100 control.
LIQUID CULTURE.
Ran a gel with: (11)
GusA P
PBBR1 GusA
Ω E PCR P
Ω PCR P
Ω PCR I
Ω E
Ω PCR I
GEL ELECTROPHORESIS PROTOCOL
Did GusA primers dissolution for PCR.
GusA PCR
[PCR PROTOCOL]
Gel Extraction by kit of lanes 3 and 5 LIQUID CULTURE.
PCR omega P, PCR omega I, PCR AraC P, PCR AraC I
Add 10 μl of each primer (LW and UP).
Add 3 μl of plasmid (P).
Add 30.4 μl buffer.
Add 5 μl Mg.
Add 8 μl DNTp’s.
Add 42.6μl H2O miliQ.
Add 1 μl RTTG polymerase.
Centrifuge (spin) 8 secs.
Add vegetable oil till the eppendorf is full.
Place eppendorf 1 mL in thermocycler.
Run PCR with program “BERNA”.
-
07/10/12
•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times) DIGESTIONS. (11.2)
07/11/12
•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X LIQUID CULTURE.
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation LIGATION PROTOCOL.
-
07/12/12
•Ran a gel with yesterday’s digestions: (12)
GEL ELECTROPHORESIS PROTOCOL .
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes TRANSFORMATION PROTOCOL.
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control GLYCEROL PROTOCOL .
•Ligated GusA PCR with B0014 E,X desphophorylated LIGATION PROTOCOL.
•Digested Pfrc54 (A3) with S,P LIQUID CULTURE.
•Desphophorylated Ω+AmyE 3’ E,X [DESPHOPHORYLATION PROTOCOL].
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07/13/12
•Ran gel with pfrc54 S,P GEL ELECTROPHORESIS PROTOCOL . (13)
•Transformation of GusA PCR + B0014 ligation TRANSFORMATION PROTOCOL.
•Transformed with GFP E0040 psBIA2.
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night TRANSFORMATION PROTOCOL.
•B. Subtitils competent cells [B.Subtilis group protocol].
07/14/12
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes:
•Extracted pellets
•Extracted plasmids [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Ran gel GEL ELECTROPHORESIS PROTOCOL .
•From transformed DH5α km 30:
GusA+B0014 DH5α Km50 24 pellets
E0040 DH5α LB Amp100
From these two we:
•Did liquid cultures LIQUID CULTURE.
•Extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid LIQUID CULTURE.
•Ran gel with this transformation GEL ELECTROPHORESIS PROTOCOL .
07/16/12
•Digested E,P AraC+ Ω+AmyE 3’
•Ω+AmyE 3’ E,P LIQUID CULTURE.
-
07/17/12
•Ran gel with yesterday’s digestions LIQUID CULTURE. (14)
•The gel we ran didn’t work, probably because the agarose was not prepared correctly.
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S LIQUID CULTURE.
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07/18/12
•From yesterday’s digestions we ran a gel GEL ELECTROPHORESIS PROTOCOL . (15)
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 LIQUID CULTURE.
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ LIGATION PROTOCOL.
•We digested 1 + Ω+AmyE 3’ E,P LIQUID CULTURE.
07/19/12
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes LIQUID CULTURE.
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E TRANSFORMATION PROTOCOL.
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P LIQUID CULTURE.
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07/20/12
•Ran gel with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL . (16)
•Transformed GusA+B0014 in two tubes TRANSFORMATION PROTOCOL.
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 LIQUID CULTURE.
07/23/12
•From 24 GusA+B0014 tubes (-) we didn’t do anything.
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P LIQUID CULTURE.
07/24/12
•Digested B0014 E with X B0014 X with E
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.
07/25/12
•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.
•Joined B0014 E with X B0014 X with E digestions.
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid LIQUID CULTURE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. Transformed TRANSFORMATION PROTOCOL.
•From LasR DH5α make liquid cultures and plate LIQUID CULTURE.
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P LIQUID CULTURE.
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated LIGATION PROTOCOL.
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07/26/12
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria LIQUID CULTURE.
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Due to problems with the way we did the transformations of ligations we repeated them:
GusAPCR X,P+ B0079 S,P dephosphorylated
GusAPCR X,P+ B0079 S,P dephosphorylated (-)
GusAPCR X,P+ A3 S,P dephosphorylated
GusAPCR X,P+ A3 S,P dephosphorylated (-)
TRANSFORMATION PROTOCOL.
•Did the following digestions LIQUID CULTURE.
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4
•K143002 X,P
•AraC+ Ω S,P
•C0179 X,S
07/27/12
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P LIGATION PROTOCOL.
07/30/12
•Transformed with ligations:
•AraC+ Ω dephosphprylated +K143002 X,P
•GusAPCR X,P+ A3 S,P dephosphorylated
•GusAPCR X,P+ B0079 S,P dephosphorylated
•Transformed the following sythesis:
•91996 Pveg 140 bp
•91997 ArsR-CzrA_promoter 1 194 bp
•91998 ArsR-CzrA_promoter 2 221 bp
•91999 ArsR-CzrA_promoter 3 213 bp
•92000 pBad-pXyl 387 bp
•92001 XylR 1117pb
•92002 CI_pro_(NAND_INHIBITOR) 774
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked. TRANSFORMATION PROTOCOL.
•Make liquid cultures of the following transformations for tomorrow LIQUID CULTURE:
•AraC+ Ω+K143002
•GusA+A3
•GusA+B0079
•Synthesis
07/31/12
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
AUGUST
08/01/12
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P LIQUID CULTURE.
-
08/02/12
•Extracted plasmids from liquid cultures[PLASMID EXTRACTION (“soft” lysis) PROTOCOL]:
AraC+ Ω+K143002
•GusA+A3
•GusA+BBR1
PCR GusA, GusA I, PCR GusA P, PCR GusA I
•Add 10 μl of each primer (LW and UP).
•Add 3 μl of plasmid (P).
•Add 30.4 μl buffer.
•Add 5 μl Mg.
•Add 8 μl DNTp’s.
•Add 42.6μl H2O miliQ.
•Add 1 μl RTTG polymerase.
•Centrifuge (spin) 8 secs.
•Add mineral oil till the eppendorf is full.
•Place eppendorf 1 mL in thermocycler.
•Run PCR with program “BERNA”.
-
03/08/12
• Ran a gel with: (18)
GEL ELECTROPHORESIS PROTOCOL .
•Ran another gel to extract with:
1.GusA PCR 1
2.GusA PCR 2
3.00
4.01
GEL ELECTROPHORESIS PROTOCOL .
•Did the following digestions LIQUID CULTURE:
• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P
•Extracted GusAPCR X,P digestion LIQUID CULTURE.
•Left the following ligation: AraC+ Ω+K143002 LIGATION PROTOCOL.
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08/06/12
•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) LIQUID CULTURE.
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated LIGATION PROTOCOL.
•Ran a Gel with: (19)
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08/08/12
BBa_B0040 6I psB1A2 Amp+ plate 1
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P LIQUID CULTURE. (19.1)
08/09/12
•From yesterday’s PCR’s :
• E0040 plasmid 1
E0040 plasmid 2
E0040 digested 1
E0040 digested 2
We purified with PCR kit
•Ran a gel GEL ELECTROPHORESIS PROTOCOL .
•Made 14 PCR’s from AraC+ Ω+AmyE 3’.
•From B0079+GusA ligation and B0040 transformation: •Grew colonies.
•Made liquid cultures LIQUID CULTURE.
•Streaked these in a new plate.
•Diluted plasmid E0080 2 1/50.
08/10/12
•Yesterday’s gel did not come out as expected so we repeated the PCR.
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08/13/12
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) GEL ELECTROPHORESIS PROTOCOL : (20)
•Extracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P LIGATION PROTOCOL.
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.
• We ran a gel to extract.
-
08/16/12
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (22)PROTOCOL]:
•Digested:
ArSR-CzrA 97 with S,P
ArSR-CzrA 98 with S,P
ArSR-CzrA 99 with S,P
E0040 PCR with E,S
Digestion Protocol.
08/20/12
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P LIGATION PROTOCOL.
08/21/12
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL].
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC LIQUID CULTURE.
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08/22/12
•We did 2 PCR’s for A3 [PCR PROTOCOL].
•Digested K143001+PBad, pXyl with E,S
LIQUID CULTURE. (22.1)
GEL ELECTROPHORESIS PROTOCOL .
•Did band extraction of:
1. K143001+PBad, pXyl E,S 10
2.K143001+PBad, pXyl E,S 11
3.K143001+PBad, pXyl E,S 12
LIQUID CULTURE.
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ LIQUID CULTURE
GLYCEROL PROTOCOL .
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ GLYCEROL PROTOCOL .
•Transformed ligations:
B0079+GusA Amp+
Pveg+XylR Chloramphenicol+ TRANSFORMATION PROTOCOL..
-
08/23/12
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this.
•Ran gel with GEL ELECTROPHORESIS PROTOCOL : (23.01)
Digestion Protocol.
08/24/12
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation.
Repeated ligation B0079+GusA.
Ligated E0040 PCR E,S+B0014 E,X dephospohylated LIGATION PROTOCOL.
Made 2 new plates from Pveg+XylR ligation and liquid cultures LIQUID CULTURE.
08/25/12
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
Ligated B0079+GusA and B0040+B0014 LIGATION PROTOCOL.
08/28/12
Made liquid cultures from transformations tht were left overnight -R0079+GusA
-R0079+GusA (-)
-E0040+B0014
-E0040+B0014 (-)
LIQUID CULTURE.
-
08/29/12
Digested Pveg+XylR 2,3,4 with E,S.
Digested Ω+AmyE 3’ with S,P DIGESTION.
Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL].
Ran a gel with
GEL ELECTROPHORESIS PROTOCOL : (23.01)
-
08/30/12
•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (24)
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S LIQUID CULTURE.
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-
-
08/31/12
•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (25)
•Dephosphorylated [DEPHOSPHORYLATION PROTOCOL]:
Ω+AmyE 3’ S,P
Ω+AmyE 3’ II S,P
6 AraC+ Ω+AmyE 3’ E,X
8 AraC+ Ω+AmyE 3’ E,X
11 AraC+ Ω+AmyE 3’ E,X
12 AraC+ Ω+AmyE 3’ E,X
•Ran a gel with A3 PCR GEL ELECTROPHORESIS PROTOCOL : (26)
•It was the second time we didn’t obtain a band from A3’s PCR.
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification.
•Ran a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL]
Gen extraction protocol.
•Did an A3 PCR [PCR PROTOCOL].
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