"
Team:Tec-Monterrey/allergen/notebook
From 2012.igem.org
(Difference between revisions)
| Line 688: | Line 688: | ||
aug_info[1] = "Stock from <i>Pichia Pastoris</i>"; | aug_info[1] = "Stock from <i>Pichia Pastoris</i>"; | ||
aug_info[2] = "Dilutions<br /><br />Re-suspension from iGem Parts experiments for the improvement of parts"; | aug_info[2] = "Dilutions<br /><br />Re-suspension from iGem Parts experiments for the improvement of parts"; | ||
| - | aug_info[3] = " | + | aug_info[3] = "<u>Transformation</u> we had a Top10 stock and it resulted that had resistance to kanamycin and chloramphenicol, turned out to be Rossetta. <br /><br /><u>MIDIPREP for pPink HC</u><br /><br /><u>Electrophoresis</u> testing to find pPink HC , good results. <br /><br />"; |
| - | aug_info[4] = " | + | aug_info[4] = "Competent cells of Top10F<br /><br />Re-suspension for Genescript’s lyophilized filter paper: nanodrop (Z maize 63 ng/ul , antihis 64 ng/ul, derf 97ng/ul) <br /><br />Purification of electrophoresis gel and quantification of nanodrop<br /><br />Transformation of iGEM parts and Genescript"; |
| - | aug_info[5] = " | + | aug_info[5] = "Dilutions<br /><br />2 Preinoculums Pichia Pastoris for electro and chemically competent cells"; |
| - | aug_info[6] = " | + | aug_info[6] = "Chemically competent cells<br /><br />Dilutions<br /><br />Phone-calling to Sponsorships"; |
| - | aug_info[7] = " | + | aug_info[7] = "Quantification of bacteria from dilutions"; |
| - | aug_info[8] = " | + | aug_info[8] = "Dilutions"; |
| - | aug_info[9] = " | + | aug_info[9] = "Transformation of His and new parts<br /><br />Quantification of bacteria from dilutions <br /><br />Development of Videogame<br /><br />Overnight Digestion<br /><br />DNA extraction from parts<br /><br />MIDIPREP for Pichia Pastoris transformation<br /><br />Stock from Pichia Pastoris"; |
| - | aug_info[10] = " | + | aug_info[10] = "Medium preparation and sterilization<br /><br />Purification of digestion<br /><br />Transformation Pichia Pastoris with HIS<br /><br />Colony selection for GFP<br /><br />Electrophoresis of yesterday’s parts and digestion"; |
| - | aug_info[11] = " | + | aug_info[11] = "Miniprep for His, BEE, GFP and AFP<br /><br />Digestion with AFIII of all of them<br /><br />Electrophoresis didn’t show results<br /><br />Stocks (His, BEE, GFP and AFP)"; |
| - | aug_info[12] = " | + | aug_info[12] = "Miniprep of His, BEE, GFP, and AFP<br /><br />Confirmation by Electrophoresis gel<br /><br />Preinoculum<br /><br />Dilutions"; |
| - | aug_info[13] = " | + | aug_info[13] = "Workshop #1 What is the material and equipment of a Lab and how to use it? <br /><br />Quantification of Bacteria (Dilutions) <br /><br />MIDIPREP <br /><br />Overnight Digestion for BEE and GFP"; |
| - | aug_info[14] = " | + | aug_info[14] = "Selling Chocolates for funding<br /><br />Meet Sponsors from Accura<br /><br />Electrophoresis of Overnight digestion (BEE and GFP) <br /><br />Purification of DNA<br /><br />Transformation of derf in Rossetta<br /><br />Dilutions"; |
| - | aug_info[15] = " | + | aug_info[15] = "Miniprep of derf, GFP and HIS<br /><br />Electrophoresis<br /><br />MIDIPREP<br /><br />Screening for transformed Rossetta<br /><br />Rossetta’s induction with IPTG<br /><br />AFP induction with Arabinose<br /><br />Stock from AFP, AFP2, HIS<br /><br />Quantification of bacteria (from dilutions)"; |
| - | aug_info[16] = " | + | aug_info[16] = "Digestion GFPS and HIS<br /><br />Electrophoresis<br /><br />MIDIPREP for HIS<br /><br />Sponsor meetings<br /><br />Transformation Rosseta with DERF (since no results)"; |
| - | aug_info[17] = " | + | aug_info[17] = "Nanodrop from the MIDIPREP<br /><br />Electrophoresis confirming GFP, AntiHIS and HIS<br /><br />Digestion<br /><br />Sterilization<br /><br />Preparation of Arabinose<br /><br />Precipitation by ETOH<br /><br />Transformation of Pichia Pastoris<br /><br />"; |
aug_info[18] = "None"; | aug_info[18] = "None"; | ||
| - | aug_info[19] = " | + | aug_info[19] = "Dilutions<br /><br />Preinoculum of TOP10F for miniprep"; |
| - | aug_info[20] = " | + | aug_info[20] = "Workshop #2 (August 21st, 2012): Bacterial DNA: Purification of Plasmid DNA (Miniprep) <br /><br />Quantification of Bacteria (Dilutions) <br /><br />Pichia pastoris preinoculum<br /><br />Preparation of SDS page<br /><br />Preparation of Staining Protocol solutions"; |
| - | aug_info[21] = " | + | aug_info[21] = "Pichia pastoris transformed growing in Minimum Media<br /><br />Quantification of induction<br /><br />SDS page wash and revealed, no results"; |
| - | aug_info[22] = " | + | aug_info[22] = "Quantification<br /><br />Pichia Induction<br /><br />SDS-Page for DERF and AFP<br /><br />Miniprep DERF and AFP<br /><br />Results for AFP protein<br /><br />Dilutions"; |
| - | aug_info[23] = " | + | aug_info[23] = "Induction<br /><br />Quantification of Bacteria (Dilutions)"; |
| - | aug_info[24] = " | + | aug_info[24] = "Transformation Rossetta with AFP<br /><br />Induction for E.Coli and Pichia Pastoris (DERF, AFP) (DERF, Zea Mays, GFP, HIS)"; |
| - | aug_info[25] = " | + | aug_info[25] = "Made Buffers (sample and lysis) <br /><br />Induction of Pichia Pastoris "; |
| - | aug_info[26] = " | + | aug_info[26] = "MIDIPREP for derf and Zea Mays<br /><br />Preparation of sample (soluble e insoluble parts) <br /><br />Sample recovery"; |
| - | aug_info[27] = " | + | aug_info[27] = "Made SDS page gels (Triscine and glycine) "; |
| - | aug_info[28] = " | + | aug_info[28] = "Again Make SDS page gels (Triscine and glycine) <br /><br /> Make counting of viability of cells from AFP project"; |
| - | aug_info[29] = " | + | aug_info[29] = "Digestion of Zm, Zm2, Derf<br /><br />DNA precipitation (Derf and Zm)"; |
aug_info[30] = "None"; | aug_info[30] = "None"; | ||
aug_type[0] = 2; | aug_type[0] = 2; | ||
| - | aug_type[1] = | + | aug_type[1] = 2; |
| - | aug_type[2] = | + | aug_type[2] = 2; |
| - | aug_type[3] = | + | aug_type[3] = 2; |
| - | aug_type[4] = | + | aug_type[4] = 2; |
| - | aug_type[5] = | + | aug_type[5] = 2; |
| - | aug_type[6] = | + | aug_type[6] = 2; |
| - | aug_type[7] = | + | aug_type[7] = 2; |
| - | aug_type[8] = | + | aug_type[8] = 2; |
| - | aug_type[9] = | + | aug_type[9] = 1; |
| - | aug_type[10] = | + | aug_type[10] = 2; |
| - | aug_type[11] = | + | aug_type[11] = 2; |
| - | aug_type[12] = | + | aug_type[12] = 2; |
| - | aug_type[13] = | + | aug_type[13] = 3; |
| - | aug_type[14] = | + | aug_type[14] = 1; |
| - | aug_type[15] = | + | aug_type[15] = 2; |
| - | aug_type[16] = | + | aug_type[16] = 2; |
| - | aug_type[17] = | + | aug_type[17] = 2; |
aug_type[18] = 0; | aug_type[18] = 0; | ||
| - | aug_type[19] = | + | aug_type[19] = 2; |
aug_type[20] = 0; | aug_type[20] = 0; | ||
| - | aug_type[21] = | + | aug_type[21] = 2; |
| - | aug_type[22] = | + | aug_type[22] = 2; |
| - | aug_type[23] = | + | aug_type[23] = 2; |
| - | aug_type[24] = | + | aug_type[24] = 2; |
| - | aug_type[25] = | + | aug_type[25] = 2; |
| - | aug_type[26] = | + | aug_type[26] = 2; |
| - | aug_type[27] = | + | aug_type[27] = 2; |
| - | aug_type[28] = | + | aug_type[28] = 2; |
| - | aug_type[29] = | + | aug_type[29] = 2; |
aug_type[30] = 0; | aug_type[30] = 0; | ||
| Line 758: | Line 758: | ||
sep_info[6] = "None"; | sep_info[6] = "None"; | ||
sep_info[7] = "None"; | sep_info[7] = "None"; | ||
| - | sep_info[8] = " | + | sep_info[8] = "Preinoculum of pichia pastoris (for transform Zm and Derf) <br /><br />Preinoculum for miniprep of GFP, His, Apim6, Derf, Zm<br /><br />Talk with Dr. Guy Cardinau <br /><br />Extraction of E. coli samples (48°C and 72°C)"; |
| - | sep_info[9] = " | + | sep_info[9] = "Workshop #3 (September 13th, 2012): Karyotype and Chromosomes"; |
| - | sep_info[10] = " | + | sep_info[10] = "<u>SDS Preparation</u><br />Sample Preparation, Place gels in the supports and loading samples<br />Washing and staining gels (5) <br /><br /><u>DNA Preparation</u><br />Miniprep crop (Zea mays 2, 2 Derf) <br />Digestion corroborating (AflII) <br />Electrophoresis<br /><br /><u>Transformation</u> <br />Screening with digestion (EcoRI) and electrophoresis (transformation went wrong) <br /><br /><u>Pichia Pastoris Transformation</u><br />Inoculate virgin Pichia in YPD and incubate @ 30 ° C<br /><br /><u>Comprobatory Induction</u><br />Harvest total of 48 h of BL21-JM109-Ctrl ctrl &<br />Inoculation BBGM in 12 tubes with minimal media cultures bites GFP colonies, His & Apim6<br />Incubation @ 30 ° C<br /><br /><u>Activity for AFP</u><br />AFP pre-inoculum of TOP10, BL21 AFP AFP & JM109<br /><br /><u>Activity for GFP</u><br />Inoculation of two tubes with colonies BBGM GFP bites -Incubation @ 30 ° C<br /><br />"; |
| - | sep_info[11] = " | + | sep_info[11] = "<u>SDS Preparation</u><br />Fade washing gels with 15 minutes. <br />Reveal gels (results were not conclusive) <br /><br /><u>DNA Preparation</u><br />Total DNA digestion overnight (Zea mays & Derf) with AflII<br /><u>Transformation</u><br />Transformation of BL21 & JM109 with Derf (transformation went wrong again) <br /><br />Xtractor of BL21-Ctrl-AFP BL21, JM109 & JM109-Ctrl-AFP (every 48 h) <br /><br /><u>Comprobatory Induction</u><br />Induction of bacteria (arabinose and NaCl) <br /><br />"; |
| - | sep_info[12] = " | + | sep_info[12] = "<u>DNA Preparation</u><br />Corroborating Electrophoresis (decide whether it can be used to transform Pichia) (samples went wrong) <br />Inoculation for Miniprep of Zea mays & Derf<br /><br /><u>Transformation</u><br />Transformation of BL21 & JM109 with reliable DNA Derf <br />Inoculate virgin Pichia in YPD and incubate @ 30 ° C<br /><br /><u>Prepare SDS-PAGE gels</u><br />Mount gels and load samples (also RosDerf, TOP10 AFP). <br /> Prepare Samples with Sample Buffer<br />Wash, fixed and stained gels overnight<br /><br /><u>Induction of AFP</u><br />(TOP10, JM109, BL21) for SDS-PAGE and purification by column"; |
| - | sep_info[13] = " | + | sep_info[13] = "<u>Miniprep Zea mays & Derf</u><br />Digestion corroborating (EcoRI) and electrophoresis. Check that they are well labeled.Overnight digestion (There was no evidence of plasmid) <br /><br /><u>Transformation of BL21 & JM109</u> <br />obtained from Miniprep Derf (transformed with DNA was not identified) <br /><br /><u>SDS PAGE</u><br />Wash with distilled water gels. 3 cycles of 5 minutes<br />Reveal gels<br /><br /><u>Research and planning of the experiment</u><br />Prepare 100 boxes of solid LB medium counts"; |
| - | sep_info[14] = " | + | sep_info[14] = "Search reagents for silver staining<br /><br />Miniprep pPink-HC and possible Zea mays & Derf<br /><br />Digestion corroborating (HindIII) and electrophoresis. Check that they are well labeled. <br /><br />Overnight digestion (AflII) <br /><br />Transformation of BL21 & JM109 with the Miniprep Derf obtained and it has been proven (Transforms failed) <br /><br />Leave blank inoculum Pichia in YPD"; |
| - | sep_info[15] = " | + | sep_info[15] = "Electrophoresis of digestion<br /><br />DNA Precipitation<br /><br />DNA Quantification<br /><br />Transformation of BL21 & JM109 with the Miniprep Derf obtained and it has been proven (Failed) <br /><br />Pichia Measure OD (OD600 = 1-2) <br /><br />Transformation with Derf<br /><br />Incubation @ 30 ° C in minimal medium boxes"; |
| - | sep_info[16] = " | + | sep_info[16] = "Leavepre- inocule BL21 virgin & JM109<br /><br />Sterilize 2 flasks (with 50 mL of LB medium) <br /><br />Leave inoculum for Rosetta miniprep Derf<br /><br />Total Harvest cultures (5 mL). Centrifuge samples and store the supernatant and pellet separately. <br /><br />Induction of bacteria (arabinose and NaCl) in fresh LB medium (not grew preinóculos) <br /><br />Total Harvest cultures (5 mL). Centrifuge samples and store the supernatant and pellet separately. <br /><br />Total Harvest cultures of JM109<br /><br />Centrifuge samples and storing pellets. Make sample Xtractor<br /><br />Induction of TOP10 & BL21 virgins (0.4% & 0.6 M NaCl arabinose) <br /><br />Sample processing, and testing laboratories contacts necessary<br /><br />"; |
| - | sep_info[17] = " | + | sep_info[17] = "<ul><li>Workshop #4 Restriction Enzymes and Electrophoresis</li><li>BL21 competent cells JM109 (Failed) </li><li>Check that we have to transform Derf</li><li>Leave cell preinoculum virgin BL21 & JM109</li><li>Induction evidencing Pichia with Zea mays & Derf</li><li>Total Harvest cultures (5 mL). Centrifuge samples and store the supernatant and pellet separately. </li><li>Concentrate samples with filters of 10 kDa</li><li>Prepare and run one gel for SDS-PAGE (12% Tricine) for GFP, His & Ctrl</li><li>Wash, fixed and stained gels overnight</li><li>Growth of bacterial cultures of 0.1 to 0.6</li><li>Induction of bacteria (arabinose and NaCl) into fresh LB medium (Failed) </li><li>Leave preinoculum of TOP10 & JM109 (AFP & Ctrl) </li><li>Total Harvest cultures (5 mL). Centrifuge samples and store the supernatant and pellet separately. </li><li>Concentrate samples with filters of 10 kDa</li><li>Prepare and run one gel for SDS-PAGE (12% Tricine) for GFP, His & Ctrl</li><li>Wash, fixed and stained gels overnight</li><li>Harvest cultures partial BL21 & TOP10</li><li>Centrifuge samples and store pellets</li></ul>"; |
| - | sep_info[18] = " | + | sep_info[18] = "<ul><li>Make silver staining</li><li>Reveal gels</li><li>Derf induction (Rosetta, JM109, BL21) for SDS-PAGE and purification by column</li><li>Growing crops (BL21 & JM109) in flasks OD600 = 0.1 to 0.6</li><li>Prepare & JM109 competent BL21</li><li>Transform JM109 & BL21 with Derf</li><li>Inoculate YPD medium with Pichia virgin Zea mays</li><li>Search DNA of Zea mays and let inocula for minipreps</li><li>Derf transformation colonies (6 each) and inoculating tubes MMBG (20 mL) </li><li>Wash with distilled water gels. Cycles 5 minutes. reveal gels</li><li>Apply this silver stain gel</li><li>Growth of bacterial cultures of 0.1 to 0.6</li><li>Induction of bacteria (arabinose and NaCl) into fresh LB medium</li><li>Wash with distilled water gels. 3 cycles of 5 minutes</li><li>Reveal gels</li><li>Xtractor lysing pellets by using lyticase. Soluble and insoluble phase store @ -20 ° C</li><li>Total Harvest culture BL21 & TOP10</li><li>Centrifuge samples and store pellets</li></ul>"; |
| - | sep_info[19] = " | + | sep_info[19] = "Workshop #5 Applications of Restriction Enzymes and Electrophoresis<br /><br />Since the crops are growing colonies transformation chopped (cell below), leaving six tubes inocula in 15 mL of LB (Rosetta, BL21, JM109 - all Derf & ctrl) <br /<br />Chop colonies and incubate in LB (Amp) <br /><br />Inoculate tubes with 15 mL of fresh LB medium (Amp) with the above culture to Miniprep<br /><br />Inoculate tubes of minimal medium from the cultures (Derf) induction of BBGM<br /><br />Miniprep DNA digestion and Zea mays. (Lack electrophoresis)"; |
| - | sep_info[20] = " | + | sep_info[20] = "Growth of bacteria in 100 mL flasks with LB (50 mL controls), 0.1-0.6<br /><br />Induction with 0.5 mM IPTG<br /><br />Centrifuge cells and move to new LB medium (100 mL or 50 mL) <br /><br />Incubation @ 16 ° C overnight<br /><br />Miniprep chopped colonies<br /><br />Corroborating digestion (EcoRI) <br /><br />Electrophoresis<br /><br />Stocks of colonies transformed with Derf<br /><br />Make glycerol stocks with 20% Derf<br /><br />Transform Pichia with Zea mays<br /><br />Incubation @ 30 ° C in minimal medium boxes<br /><br />Pass Medium crops methanol (5 ml per tube) <br /><br />"; |
| - | sep_info[21] = " | + | sep_info[21] = "Total Harvest cultures (20 mL) <br /><br />Centrifuge samples and store the supernatant and pellet separately<br /><br />Xtractor lysing pellets by using lyticase. Soluble and insoluble phase store @ -20 ° C<br /><br />Preparing samples for purification material and columns of the His-tag protein"; |
| - | sep_info[22] = " | + | sep_info[22] = "Prepare Samples with Sample Buffer<br /><br />Prepare SDS-PAGE gels<br /><br />Mount gels and load samples. Running @ 90 V, 2.00 A<br /><br />Wash, fixed and stained gels overnight<br /><br />Purify His-tag protein by columns. Analyze results"; |
| - | sep_info[23] = " | + | sep_info[23] = "Made execution of AFP experiment <br /><br />Made SDS gels <br /><br />Preparing samples for purification material and columns of the His-tag protein"; |
sep_info[24] = "None"; | sep_info[24] = "None"; | ||
| - | sep_info[25] = " | + | sep_info[25] = "Wiki Freeze<br /><br />Activity assay for anti-IgE and anti-His, also activity assay for shuttle sequence in Pichia pink"; |
sep_info[26] = "None"; | sep_info[26] = "None"; | ||
sep_info[27] = "None"; | sep_info[27] = "None"; | ||
| Line 789: | Line 789: | ||
sep_type[6] = 0; | sep_type[6] = 0; | ||
sep_type[7] = 0; | sep_type[7] = 0; | ||
| - | sep_type[8] = | + | sep_type[8] = 2; |
| - | sep_type[9] = | + | sep_type[9] = 3; |
| - | sep_type[10] = | + | sep_type[10] = 2; |
| - | sep_type[11] = | + | sep_type[11] = 2; |
| - | sep_type[12] = | + | sep_type[12] = 2; |
| - | sep_type[13] = | + | sep_type[13] = 2; |
| - | sep_type[14] = | + | sep_type[14] = 2; |
| - | sep_type[15] = | + | sep_type[15] = 2; |
| - | sep_type[16] = | + | sep_type[16] = 3; |
| - | sep_type[17] = | + | sep_type[17] = 2; |
| - | sep_type[18] = | + | sep_type[18] = 2; |
| - | sep_type[19] = | + | sep_type[19] = 3; |
| - | sep_type[20] = | + | sep_type[20] = 2; |
| - | sep_type[21] = | + | sep_type[21] = 2; |
| - | sep_type[22] = | + | sep_type[22] = 2; |
| - | sep_type[23] = | + | sep_type[23] = 2; |
sep_type[24] = 0; | sep_type[24] = 0; | ||
| - | sep_type[25] = | + | sep_type[25] = 3; |
sep_type[26] = 0; | sep_type[26] = 0; | ||
sep_type[27] = 0; | sep_type[27] = 0; | ||
Revision as of 03:51, 27 September 2012
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