Team:University of Lethbridge/Notebook/Project4October

Back to The University of Lethbridge Main Notebook

Jaden
Objective: Grow up cells containing the xylE gene from the glycerol stocks and plates (Selina had transformed with DH5a) in LB + Amp liquid media. The gene was sent to us in pVL1392 (modified pUC8 for viral expression, with Amp resistance).

Subcultured four tubes: Two from glycerol stocks and two from colonies on a plate.

Christa, Munima
Objective: Plasmid prep the xylE gene that Jaden subcultured yesterday.

Christa: Plasmid prepped one subculture tube from 'glycerol stock' and 'plate' using the Eppendorf FastPlasmid Mini Prep Kit. Stored 50 uL aliquots in the -20 C iGEM freezer box (iGEM Plasmids) labeled "xylE from glycerol stock" and "xylE from plate", and have a big blue "P" or "G" on the top, respectively.

Munima: Plasmid prepped one subculture tube from 'glycerol stock' and 'plate' using the QiaPrep Spin MiniPrep Kit. Plasmids are stored in 50 uL aliquots in the -20 C iGEM freezer in the iGEM Plasmids box. They are labeled "xylE plasmid; plate 2; Oct. 7/08" and "xylE plasmid; glycerol 2; Oct. 7/08".

Christa, Roxanne
Ran gel to assess if gel extractions and plasmid preps worked. See gel for results. Plasmid preps of xylE yielded no results with the Eppendorf Kit, so use Qiagen kit from now on.

Roxanne
Objective: Set up PCR for xylE gene with Phusion enzyme.

Master Mix for 5 reactions with Phusion: - 10x Buffer: 25 uL - 10 mM dNTPs: 5 uL - 50 mM Mg2+: 7.5 uL - 10 uM RF: 5 uL - 10 uM RR: 5 uL - Phusion polymerase: 1 uL - H20: 196.5 uL - Total volume is 245 uL. Therefore, 49.0 uL in each reaction tube. - template: 1 uL into each reaction tube to equal 50 uL volume

Cycle conditions: 1. Denaturation: 94 C for 1 min 2. a. Denaturation: 94 C for 30 seconds b. Annealing: 52 C for 30 seconds c. Extension: 70 C for 30 seconds Repeat Step 2 for 30 cycles 3. Final extension: 72 C for 10 minutes, then hold at 4 C.

Munima
Objective: Subculture cells from transformation to determine if it was successful

Inoculated three colonies from transformation done yesterday (by Roxanne) of DH5-alpha + pE43 into LB + Tet (100 ug/mL). Tubes were put into the shaker incubator at 37 C overnight.

October 11, 2008
-Ran gel of the restriction digested xylE amplicon and the pE43 plasmid prep.

-PCR of the ohbA, ohbB, ohbC, ohbR genes from pE43 with an annealing temperature of 51.0 C.

-Ran a gel of the ohb gene amplicons

-Restriction digested the ohb genes with XbaI and PstI overnight at 37.0C

Roxanne
-Ran a gel of the PCR products to determine the result of the PCR reaction.



-Set up a Restriction Digest of the xylE gene and pSB1A2 for GFP sub, using XbaI and PstI. Incubate overnight at 37.0C.

Back to The University of Lethbridge Main Notebook

Roxanne et al
-Ohb operon PCR using Phusion polymerase (4 x 50 uL reactions):

Master Mix (1x): -5x Phusion buffer: 10 uL -10 mM dNTP: 1 uL -10 mM forward primer: 1 uL -10 mM reverse primer: 1uL -Water: 35.5 uL -Phusion polymerase: 0.5 uL -Template: 1 uL

Ligation of xylE into pSB1A2:

[pSB1A2] = 25 ng/uL; 2079 bp

[xylE] = 30 ng/uL; 924 bp

ohbA = 530 bp; ohbB = 1286 bp; ohbC = 1352 bp; ohbR = 716 bp

Calculation: -amount of xylE (ng) to add = [(bp insert)(vector concentration)]/(bp vector) * 3 = 33.3 ng

Ligation Reaction: -10x Buffer: 2 uL -50% PEG4000: 2 uL -T4 DNA Ligase: 0.33 uL -pSB1A2: 7 uL -xylE: 8 uL -optimal water: 0.67 uL

Roxanne
Objective: PCR of ohb operon using Phusion polymerase (3 x 20 uL reaction)

Master Mix (1x): -5x Phusion HF Buffer: 4 uL -10 mM dNTP: 0.4 uL -10 mM forward primer: 0.4 uL -10 mM reverse primer: 0.4 uL -optimal water: 13.6 uL -template: 1 uL

Objective: Restriction digest of pSB1A2

Reaction: -Tango Buffer: 4 uL -Template: 30 uL -Enzyme 1 (XbaI): 2 uL -Enzyme 2 (PstI or SpeI): 2 uL -d2H2O: 2 uL

SpeI is required for CheZ ligation.

PstI is required for ohb gene ligations.

- Transformed ligation products into DH5alpha. Plated 25 uL on LB + Amp (100 ug/mL).

Munima, Roxanne
Objective: Assess results of ligation/transformation performed yesterday of xylE + pSB1A2 into DH5alpha.

- 1 colony

Colony PCR of xylE with Taq polymerase - to assess if insert is present in ligation product (2 x 10 uL reaction) Master Mix: - 10x Buffer: 1 uL -10 mM dNTP: 0.2 uL -50 mM MgCl2: 0.3 uL -10 mM forward primer: 0.25 uL -10 mM reverse primer: 0.25 uL -d2H2O: 7.0 uL -Taq polymerase: 0.5 uL -template: 1 uL

PCR conditions: 1. Initial denaturation: 95 C for 15 mins 2. a. Denaturation: 94 C for 30 seconds b. Annealing: 56 C for 30 seconds c. Extension: 68 C for 60 seconds Repeat step 2 for 35 cycles 3. Final extension: 68 C for 60 seconds. 4. Hold at 4 C

- Ran a gel and did gel extraction of pzero+CheZ, and pSB1A2

Roxanne
The chemical competency plates don't have much growth. -1 colony on DH5a + pUC19 -8 colonies on BL21 DE3 + pUC19 -1 speck on RP1616 + pUC19

Obviously, the transformation didn't go too well. We will redo it with two sets of RP1616 cells, 1 BL21 and 1 DH5a.

The colony PCR of xylE had weird results on the gel. The subculture was plasmid prepped and run on a gel. We will redo a screening PCR of this if the size looks good. -dephosphorylate pSB1A2 (XbaI, SpeI) -then ligate CheZ into it -ligate pLacI + TetR -re-PCR ohb B, C and R (with higher annealing temperature) -re-PCR xylE in pSB1A2 with VF2, VR and xylE primers -setup restriction digest of ohbA with XbaI and PstI, and pSB1A2 with EcoRI and SpeI; ng ChZ needed = 55.34; put 4.45 uL of CheZ into 1 uL of pSB1A2

Munima
Objective: re-do the xylE PCR with Taq polymerase

Master Mix (2 x 10 uL reaction) -10x Buffer: 1 uL -10 mM dNTP: 0.2 uL -50 mM MgCl2: 0.3 uL -10 mM forward primer: 0.2 uL -10 mM reverse primer: 0.2 uL -Taq polymerase: 0.5 uL -d2H2O: 7.0 uL -template: 0.6 uL

-Make a master mix for two different primer sets

Restriction digest ohbA with Xba1 and PstI; pSB1A2 with EcoRI and SpeI.

Roxanne
Transformation of the overnight ligation of pLacI and TetRsub and CheZ + pSB1A2 were both good.

The gel of the xylE screening PCR had good results.

-gel extraction of ohbA (XbaI, PstI) and pSB1A2 (EcoRI and SpeI) -quantification from the gel: [pSB1A7] = 105 ng/uL; [ohbA] = 10 ng/uL

Roxanne
Objective: Screening PCR of CheZ and TetR complete colonies using VF2/VR (forward and reverse primers) and Econo Taq polymerase

Master Mix (1 x; did 7 x 10 uL reactions): -10x Buffer: 1 uL -10 mM dNTP: 0.2 uL -50 mM MgCl2: 0.3 uL -10 mM VF2: 0.2 uL -10 mM VR: 0.2 uL -Taq polymerase: 0.5 uL -d2H20: 7.0 uL -template: 0.6 uL

Screening PCR of CheZ using CheZ primers and Taq polymerase:

Master Mix (1 x; did 4 x 10 uL reactions): -10x Buffer: 1 uL -10 mM dNTP: 0.2 uL -50 mM MgCl2: 0.3 uL -10 mM VF2: 0.2 uL -10 mM VR: 0.2 uL -Taq polymerase: 0.5 uL -d2H20: 7.0 uL -template: 0.6 uL

Ohb PCR with Phusion polymerase:

Master Mix (1x; did 3 x 20 uL reaction): -5x Phusion HF Buffer: 4 uL -10 mM dNTP: 0.4 uL -10 mM forward primer: 0.4 uL -10 mM reverse primer: 0.4 uL -optimal water: 13.6 uL -template: 1 uL

Ligation of OhbA into pSB1A2 -used 5.53 uL of insert/uL of vector -[ohbA] = 65.0 ng

Reaction (20 uL): -ohbA: 8.3 uL -pSB1A2: 1.5 uL -buffer: 1.5 uL -d2H2O: 3.0 uL -T4 DNA Ligase: 0.2 uL

Roxanne
Objective: PCR using the screening primers on CheZ + pSB1A2 and TetR complete

Same protocol as on October 20, 2008.

Roxanne
Objective: Ligation of ohbA and ohbC into pSB1A2

From gel: [ohbC] = 10 ng/uL gel extraction product; [ohbA] = 108 ng/uL

Using the calculation from October 15, 2008, for ligation: -12.681 uL insert/uL vector for ohbC -0.46 uL insert/uL vector for ohbA

For procedure: - Used 13 uL of ohbC for 1 uL of pSB1A2 and 1 uL of ohbA for 2 uL of pSB1A2.

-Sequencing results were obtained for xylE.

Roxanne
-Transformation of ohbA and ohbC plasmids into DH5a.

Roxanne
-We have colonies!!!

-picked 3 of each and subcultured into LB + amp media. Incubated overnight at 37.0 C

Roxanne
-Plasmid prepped the ohbA and ohbC plasmids.

-Performed a screening PCR on the ohbA and ohbC genes using VF2/VR and the primers for the individual genes.

-ohbC came up a little short. Oops, forgot that there was a PstI restriction site in the gene. No time to redo with mutagenesis.

-Prepared ohbA_pSB1A2, xylE_pSB1A2 and cheZ_pSB1A2 to be sent off to the registry, and prepared ohbA to be sent of for sequencing.