Team:METU Turkey/Experiments

Welcome!
We are Team METU_Turkey!

Experiments/Documentation of the development of our project


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




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

Protocols

Standard iGEM Protocols

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