Difference between revisions of "Team:METU Turkey/Experiments"
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<a class="one" href="https://2015.igem.org/Team:METU_Turkey/Safety">Safety</a> | <a class="one" href="https://2015.igem.org/Team:METU_Turkey/Safety">Safety</a> | ||
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− | <h2 align=center>Experiments</h2> | + | <h2 align=center>Experiments/Documentation of the development of our project</h2> |
− | < | + | |
− | </ | + | <pre> |
− | </div> | + | |
+ | July 15th, Wednesday | ||
+ | Stock cells were planted in empty plates | ||
+ | |||
+ | July 16th, Thursday | ||
+ | Competent cells were prepared | ||
+ | 7 strains | ||
+ | Transformation was done at night for control purposes, | ||
+ | 5 strains were negative (amp plate 4 min 4000 rpm) | ||
+ | |||
+ | July 17th, Friday | ||
+ | Kitten gene was extracted | ||
+ | 5 strains | ||
+ | Transformation was done for control purposes. | ||
+ | This was so because 5 strains were negative in the previous one. | ||
+ | Kumomax enzyme was planted from agar plate to agar plate. | ||
+ | |||
+ | July 18th, Saturday | ||
+ | All of the transformations were positive. | ||
+ | Kumomax enzyme was planted from agar to LB. | ||
+ | |||
+ | July 19th, Sunday | ||
+ | Plasmid isolation was done. | ||
+ | Digestion was done (with not1) | ||
+ | Gel electrophoresis was done, The result was positive. | ||
+ | Gel extraction was done | ||
+ | Nanodrop result was 77ng/microl | ||
+ | |||
+ | July 20th, Monday | ||
+ | Plate with zeocin prepared (salt 5g,) total 200 mL | ||
+ | Low salt medium (LB) was prepared, total 200 mL | ||
+ | |||
+ | July 21st, Tuesday | ||
+ | Transformation was done. PGapZ -alpha was duplicated. | ||
+ | |||
+ | July 22nd, Wednesday | ||
+ | PGapZ-alpha was planted from agar to LB | ||
+ | |||
+ | July 23rd, Thursday | ||
+ | Plasmid isolation was done for PGapZ-alpha | ||
+ | Restriction was done with NotI (PGapZ-alpha) | ||
+ | Gel electrophoresis was done (PgapZ-alpha), the result was positive | ||
+ | Geş extraction was done, the nanadrop result was positive | ||
+ | |||
+ | July 24th, Friday | ||
+ | Ligation was done for PGapZ-alpha-kumomax | ||
+ | Transformation was done | ||
+ | |||
+ | July 25th, Saturday | ||
+ | Transformation results were negative | ||
+ | Transformation was done | ||
+ | 323101 chl | ||
+ | 323106 chl | ||
+ | 323117 chl | ||
+ | I13504 (GFP) Amp | ||
+ | 8 transformation plates total, two plates from each. | ||
+ | |||
+ | July 26th, Sunday | ||
+ | Transformation results were positive | ||
+ | The colonies were taken from agar and planted in LB | ||
+ | |||
+ | July 27th, Monday | ||
+ | Plasmid isolation was done for the promoter in Lb and GFP | ||
+ | Restriction was done | ||
+ | For promoters, (101, 106, 117) Spe1 and pst1 | ||
+ | For GFP, Xba1 and PSt 1 | ||
+ | Gel electrophoresis ws done for the cut promoters and GFP | ||
+ | 101, 106, 117 and Gfp were again planted to LB from agar | ||
+ | |||
+ | July 28th, Tuesday | ||
+ | Plasmid isolation was done for 101, 106, 117 and GFP | ||
+ | Restriction was done | ||
+ | Gel electrophoresis was done for the cut 101, 106, 117 and GFP, | ||
+ | the results were negative | ||
+ | 101, 106, 117 and GFP were again planted in LB from agar | ||
+ | |||
+ | July 29th, Wednesday | ||
+ | Plasmid isolation was done for 101, 106, 117 and GFP | ||
+ | Restriction was done for 101, 106, 117 and GFP | ||
+ | Gel electrophoresis was done for the cut 101, 106, 117 and GFP, | ||
+ | the results were positive only for the promoters, 101, 106, and 117 | ||
+ | Gel extraction was done for promoters | ||
+ | Nanodrop results were low | ||
+ | 101, 106, 117 and GFP were again planted in LB from agar | ||
+ | |||
+ | July 30th, Thursday | ||
+ | Plasmid isolation was done for 101, 106, 117 and GFP | ||
+ | Centrifuge was lowered to 12000 rpm from 13000 rpm | ||
+ | Restriction was done for 101, 106, 117 and GFP | ||
+ | Optimization was done for restriction | ||
+ | |||
+ | <img hspace="80px" src="https://static.igem.org/mediawiki/2015/2/2b/4_sayfa_elif_tablo.PNG" width="400px"> | ||
+ | |||
+ | |||
+ | The electrophoresis results were acquired for set 2 and set 2, set 1 | ||
+ | did not produce results | ||
+ | Gel extraction was done for the 4 bands that yielded positive results | ||
+ | Nanodrop results were low | ||
+ | Plantation was done again from agar to LB | ||
+ | |||
+ | July 31st, Friday | ||
+ | Plasmid isolation as done for GFP | ||
+ | Restriction was done by Xba1 and Pst1 for GFP | ||
+ | Gel electrophoresis was done, the results were positive | ||
+ | Gel extraction was done | ||
+ | Nanodrop results were low | ||
+ | Plantation from agar to LB was done again for GFP | ||
+ | |||
+ | August 1st, Saturday | ||
+ | Plasmid isolation was done for GFP | ||
+ | |||
+ | August 3rd, Monday | ||
+ | Restriction was done for GFP | ||
+ | Gel electrophoresis was performed on the ones that were cut | ||
+ | The results were positive | ||
+ | Gel extraction was performed | ||
+ | Nanodrop results were good | ||
+ | |||
+ | August 4th, Tuesday | ||
+ | Ligation was done for promoters and GFP | ||
+ | |||
+ | August 5th, Wednesday | ||
+ | Transformation results were negative | ||
+ | Ligation was performed again | ||
+ | Transformation was done for those who had been ligated | ||
+ | |||
+ | August 6th, Thursday | ||
+ | Transformation results are negative | ||
+ | Ligation was performed again for promoters and GFP | ||
+ | Transformation was done | ||
+ | Ligation was done for PGapZ-alpha and kumomax | ||
+ | Transformation was done | ||
+ | |||
+ | August 7th, Friday | ||
+ | The transformations done for promoter-GFP and PGapZ-alpha- | ||
+ | kumomaz were negative | ||
+ | 400 mL Amp agar medium was prepared, put into plates | ||
+ | |||
+ | August 10th, Monday | ||
+ | Streak plate was done for 101, 106, 117 and GFP (8 in total) | ||
+ | Chl plates were prepared | ||
+ | -400 mL agar medium | ||
+ | -5 g salt was used instead of 10 g salt | ||
+ | |||
+ | August 11th, Tuesday | ||
+ | The streaked plated 101, 106,117 and GFP were planted in | ||
+ | LB (8 tubes in total) | ||
+ | LB was prepared (400 mL, 5 g salt) | ||
+ | |||
+ | August 12th, Wednesday | ||
+ | Plasmid isolation was done for 101, 106, 117 and GFP | ||
+ | Restriction was done for GFP | ||
+ | |||
+ | <img hspace="80px" src="https://static.igem.org/mediawiki/2015/3/3a/7_son_sayfa_elif_tablo.PNG" width="400px"> | ||
+ | </pre> | ||
+ | </div></br> | ||
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<h2 align=center>Protocols</h2> | <h2 align=center>Protocols</h2> | ||
<p align=center>Standard iGEM <a class="one" href="http://parts.igem.org/Help" target="_blank">Protocols</a></p> | <p align=center>Standard iGEM <a class="one" href="http://parts.igem.org/Help" target="_blank">Protocols</a></p> | ||
+ | <pre> | ||
+ | Transformation Protocol: | ||
+ | 1)Throw competent cell on ice | ||
+ | 2) Mix ligation product 50 microliter cell+2 microliter plasmid | ||
+ | 3)Incubate cells on ice 30 mins | ||
+ | 4) Heat shock 55 sec CaCl2, 75 sec RuCl2 | ||
+ | Add 900 ml LB | ||
+ | 5)Incubate 37 celcius for 80 minute | ||
+ | 6)Centrifuge at 3000 po m 10 mın | ||
+ | 7)Discard supernatant | ||
+ | 8)Resuspend pellet in 100 ml LB | ||
+ | 9)Spread cells and wait 14-16 hours in the incubator | ||
+ | |||
+ | |||
+ | LB Broth Protocol: | ||
+ | 1)10 gr peptone | ||
+ | 2)5 gr yeast extract | ||
+ | 3)10 gr NaCl | ||
+ | 4)up to 1000ml dH20 & autoclave | ||
+ | |||
+ | |||
+ | LB Agar Protocol: | ||
+ | 1)10 gr peptone | ||
+ | 2)5 gr yeast extract | ||
+ | 3)10 gr NaCl | ||
+ | 4)15 gr agar | ||
+ | 5)up to 1000ml dH20 & autoclave | ||
+ | 6)use 20 gr for 1lt dH2O | ||
+ | |||
+ | |||
+ | ONC Protocol: | ||
+ | 1)Put 5 ml LB into a tube. | ||
+ | 2)Put a loop of E.coli dH5 aseptic. | ||
+ | 3)Seal the tube using parafilm. | ||
+ | 4)Place the tube diagonally into a shaker for 14-18 hours.(optimum 14) | ||
+ | |||
+ | |||
+ | Competent Cell Preparation by Calcium Chloride Protocol: | ||
+ | 1)Inoculate 1ml ONC to 100 ml LB in a flask. | ||
+ | 2)Incubate the flask for 2-4h at 37 ˚C, check via spectrophotometer at | ||
+ | 600nm for 0.300.. | ||
+ | 3)Divide the solution into two falcon tubes(50 ml). | ||
+ | 4)Spin them down at +4 ˚C and 5000g for 5min. | ||
+ | 5)Discard supernatant. | ||
+ | 6)Resuspend the cells with 10 ml cold CaCl2. | ||
+ | 7)Put in ice for 10 minutes. | ||
+ | 8)Spin the suspension down at +4 ˚C, 5000g for 5 minutes. | ||
+ | 9)Discard supernatant. | ||
+ | 10)Add 10 ml cold CaCl2 and resuspend the cells. | ||
+ | 11)Put the cells in ice for 30 min. | ||
+ | 12)Centrifuge at 5000 rpm for 5 min at +4 ˚C. | ||
+ | 13)Discard supernatant. | ||
+ | 14)Put 1 ml CaCl2 and dissolve pellet. | ||
+ | 15)Put it on ice for 5 min. and keep it at +4 ˚C. | ||
+ | |||
+ | |||
+ | Getting the DNA Parts from the Kit Plate: | ||
+ | 1)Add 10 microliter of ddH2O into the wanted well. | ||
+ | 2)Wait and get the part by pipetting. | ||
+ | 3)Make sure to keep stock from these parts since there is so less of | ||
+ | them. | ||
+ | 4)In case of having too less sample, you can dilute the stock 10:1 | ||
+ | with ddH2O. | ||
+ | |||
+ | |||
+ | Midi Prep Plasmid Isolation Protocol: | ||
+ | A)Bacterial culture, harvest, and lysis. | ||
+ | 1)Pellet 25 ml (high copy) or 100ml (low copy) overnight LB culture | ||
+ | at 6000xg for 15 min at +4˚C. | ||
+ | 2)Homogeneously resuspend the bacterial pellet in 4 ml Buffer P1. | ||
+ | 3)Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4-6 times, | ||
+ | and incubate at room temperature for 5 min. | ||
+ | 4)Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4-6 times, | ||
+ | and incubate on ice for 15 min. | ||
+ | B)Bacterial lysate clearing | ||
+ | 5)Centrifuge at ≥20.000 xg for 30 min at +4 ˚C. Re-centrifuge the | ||
+ | supernatant at ≥20.000 xg for 15 min at +4 ˚C. | ||
+ | C)Bind, wash, and elute plasmid DNA on QIAGEN-tip. | ||
+ | 6)Equilibrate a QIAGEN-tip 100 by applying 4ml Buffer QBT and allow | ||
+ | column to empty by gravity flow. | ||
+ | 7)Apply the supernatant(step 5) to the QIAGEN-tip and allow it to enter | ||
+ | the resin by gravity flow. | ||
+ | 8)Wash the QIAGEN-tip with 2x10 ml Buffer QC. Allow Buffer QC to move | ||
+ | through the QIAGEN-tip by gravity flow. | ||
+ | 9)Elute DNA with 5 ml Buffer QC into clean 2 ml, 15ml or 50ml vessel. | ||
+ | D)Precipitate, wash, and redissolve plasmid DNA | ||
+ | 10)Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the | ||
+ | eluted DNA and mix. Centrifuge at ≥ 15.000 xg for 30 min at +4℃. | ||
+ | Carefully decant supernatant. | ||
+ | 11) Wash DNA pelelt with 2ml room-temperature 70% ethanol and | ||
+ | centrifuge at ≥ 15.000 xg for 10 min. Carefully decant supernatant. | ||
+ | 12) Air-dry pellet for 5-10 min and redissolve DNA in a suitable volume | ||
+ | of buffer. | ||
+ | |||
+ | |||
+ | Plasmid Isolation Protocol: | ||
+ | Resuspend the pelleted cells in 250μL of the Resuspension Solution. | ||
+ | Transfer the cell suspension to a microcentrifuge tube. The bacteria | ||
+ | should be resuspended | ||
+ | completely by vortexing or pipetting up and down until no cell clumps | ||
+ | remain. | ||
+ | Add 250μL of the Lysis Solution and mix thoroughly by inverting the | ||
+ | tube 4-6 times until the solution becomes viscous and slightly clear. | ||
+ | Add 250μL of the Lysis Solution and mix immediately and thoroughly | ||
+ | by inverting the tube 4-6 times. | ||
+ | Centrifuge for 5 min to pellet cell debris and chromosomal DNA. | ||
+ | Transfer the supernatant to the supplied GeneJET spin column by | ||
+ | decanting or pipetting. Avoid disturbing or transferring the white | ||
+ | precipitate. | ||
+ | Centrifuge for 1 min. Discard the flow-through and place column | ||
+ | back into the same collection tube. | ||
+ | Add 500μL of the Wash Solution to the GeneJET spin column. | ||
+ | Centrifuge for 30-60 seconds and discard the flow-through. Place | ||
+ | the column back into the same collection tube. | ||
+ | Repeat the wash procedure using 500μL of the Wash Solution. | ||
+ | Discard the flow-through and centrifuge for an additional 1 min to | ||
+ | remove residual Wash Solution. This step is essential to avoid residual | ||
+ | ethanol in plasmid preps.Transfer the GneJET spin column into a fresh | ||
+ | 1.5 ml microcentrifuge tube. Add 50μL of the Elution Buffer to the | ||
+ | center of GeneJET spin column membrane to elute the plasmid DNA. | ||
+ | Take care not to contact the membrane with the pipette tip. Incubate | ||
+ | for 2 min at room temperature and centrifuge for 2 min. | ||
+ | Discard the column and store the purified plasmid DNA at -20 ˚C. | ||
+ | |||
+ | |||
+ | 80% Glycerol Preparation: | ||
+ | Add 80ml 99.7% glycerol to 20ml demineralized water | ||
+ | Autoclave | ||
+ | |||
+ | </pre> | ||
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Latest revision as of 13:29, 4 October 2015
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