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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Competent Cell Preparation by Calcium Chloride Protocol: | Competent Cell Preparation by Calcium Chloride Protocol: | ||
1)Inoculate 1ml ONC to 100 ml LB in a flask. | 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.. | + | 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). | 3)Divide the solution into two falcon tubes(50 ml). | ||
4)Spin them down at +4 ˚C and 5000g for 5min. | 4)Spin them down at +4 ˚C and 5000g for 5min. | ||
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1)Add 10 microliter of ddH2O into the wanted well. | 1)Add 10 microliter of ddH2O into the wanted well. | ||
2)Wait and get the part by pipetting. | 2)Wait and get the part by pipetting. | ||
− | 3)Make sure to keep stock from these parts since there is so less of them. | + | 3)Make sure to keep stock from these parts since there is so less of |
− | 4)In case of having too less sample, you can dilute the stock 10:1 with ddH2O. | + | them. |
+ | 4)In case of having too less sample, you can dilute the stock 10:1 | ||
+ | with ddH2O. | ||
Midi Prep Plasmid Isolation Protocol: | Midi Prep Plasmid Isolation Protocol: | ||
A)Bacterial culture, harvest, and lysis. | 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. | + | 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. | 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. | + | 3)Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4-6 times, |
− | 4)Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4-6 times, and incubate on ice for 15 min. | + | 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 | 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. | + | 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. | 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. | + | 6)Equilibrate a QIAGEN-tip 100 by applying 4ml Buffer QBT and allow |
− | 7)Apply the supernatant(step 5) to the QIAGEN-tip and allow it to enter the resin by gravity flow. | + | column to empty 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. | + | 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. | 9)Elute DNA with 5 ml Buffer QC into clean 2 ml, 15ml or 50ml vessel. | ||
D)Precipitate, wash, and redissolve plasmid DNA | 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. | + | 10)Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the |
− | 11) Wash DNA pelelt with 2ml room-temperature 70% ethanol and centrifuge at ≥ 15.000 xg for 10 min. Carefully decant supernatant. | + | eluted DNA and mix. Centrifuge at ≥ 15.000 xg for 30 min at +4℃. |
− | 12) Air-dry pellet for 5-10 min and redissolve DNA in a suitable volume of buffer. | + | 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: | 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. | + | Resuspend the pelleted cells in 250μL of the Resuspension Solution. |
− | Add 250μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. | + | Transfer the cell suspension to a microcentrifuge tube. The bacteria |
− | Add 250μL of the Lysis Solution and mix immediately and thoroughly by inverting the tube 4-6 times. | + | 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. | 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. | + | Transfer the supernatant to the supplied GeneJET spin column by |
− | Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube. | + | decanting or pipetting. Avoid disturbing or transferring the white |
− | 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. | + | 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. | 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. | + | Discard the flow-through and centrifuge for an additional 1 min to |
− | 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. | + | 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. | Discard the column and store the purified plasmid DNA at -20 ˚C. | ||
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− | <img hspace="375px" align=center src="https://static.igem.org/mediawiki/2015/ | + | <img hspace="375px" align=center src="https://static.igem.org/mediawiki/2015/e/eb/Methodmetuturkey343.PNG" width="600px"> |
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</div> | </div> |
Latest revision as of 13:29, 4 October 2015
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