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SEPTEMBER: WEEK 4
September, 20th
Screening PCR on 6 colonies picked from I55 was performed. The results were the follow:
Inoculum of I52, I53, I55, I56 and I57 in LB+Amp for MiniPrep of the following day
September, 21st
Miniprep and Nanodrop quantification for:
- I52 : 92,8 ng/ul
- I53 : 118,5 ng/ul
- I55 : 121,1 ng/ul
- I56 : 159,6 ng/ul
- I57 : 103 ng/ul
For I61 the DNA is already available quantified 147,1 ng/ul.
After 3 hour digestion was performed:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 (ul) | Enzyme 2 (ul) | Buffer H (ul)
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| I52 | Vector | 25 | 10,8 | 9,7 | 1 EcoRI | 1 XbaI | 2,5
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| I53 | Vector | 25 | 8,5 | 12 | 1 EcoRI | 1 XbaI | 2,5
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| I55 | Insert | 25 | 14 | 6,5 | 1 EcoRI | 1 SpeI | 2,5
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| I56 | Insert | 25 | 10 | 10,5 | 1 XbaI | 1 PstI | 2,5
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| I57 | Vector | 25 | 9,7 | 10,8 | 1 EcoRI | 1 XbaI | 2,5
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| I61 | Vector | 25 | 6,8 | 13,7 | 1 EcoRI | 1 XbaI | 2,5
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Gel run/cut of samples
As you can see only I52, I56, I57 and I61 run correctly so gel extraction was performed on this samples:
- I52 = 29,4 ng/ul
- I56 = 15,4 ng/ul
- I57 = 24,7 ng/ul
- I61 = 30,2 ng/ul
We already had digested DNA so we could perform new ON ligations:
- I62 = I59(X-P) + I39(S-P)
- I63 = I61(E-X) + I38(E-S)
- I64 = I52(E-X) + I58(E-S)
- I65 = I39(S-P) + I56(X-P)
- I66 = I57(E-X) + I60(E-S)
Tecan Test on self-inducible promoters was performed on prepared samples with a different protocol that is to bring the cultures to a known OD.
September, 22nd
Trasformation of ligation of I62, I63, I64, I65 and I66 into E.coli TOP10.
MiniPrep and quantification of:
- MyYeast1 = 177,8 ng/ul
- MyYeast2 = 98,9 ng/ul
- MyYeast3 = 197,2 ng/ul
Screening digestion was performed as follows:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 (ul) | Enzyme 2 (ul) | Buffer Tango (ul)
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| MyYeast1 | Screening | 25 | 1,2 | 19,3 | 1 EcoRI | 1 NsiI | 2,5
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| MyYeast2 | Screening | 25 | 2 | 18,5 | 1 EcoRI | 1 NsiI | 2,5
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| MyYeast3 | Screening | 25 | 1 | 19,5 | 1 EcoRI | 1 NsiI | 2,5
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Gel run of samples was performed
Noone samples was positive
Glycerol stocks of MyYeast1, MyYeast2, MyYeast3 was performed.
Inoculum in LB+Amp+Cm12,5 from glycerol stocks and colony for antibiotic resistance testing of I47_S1, I48_S1, I49_S1 and I13522.
September, 23rd
Digestion of yesterday are repeted (because noone of them was postitive) as follows:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme (ul) | Buffer H Roche 10X (ul)
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| MyYeast1 | Screening | 25 | 1,2 | 20,3 | 1 EcoRI | 2,5
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| MyYeast2 | Screening | 25 | 2 | 19,5 | 1 EcoRI | 2,5
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| MyYeast3 | Screening | 25 | 1 | 20,5 | 1 EcoRI | 2,5
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| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme (ul) | Buffer Red (ul)
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| MyYeast1 | Screening | 25 | 1,2 | 20,3 | 1 NsiI | 2,5
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| MyYeast2 | Screening | 25 | 2 | 19,5 | 1 NsiI | 2,5
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| MyYeast3 | Screening | 25 | 1 | 20,5 | 1 NsiI | 2,5
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Gel run of samples was performed
(foto)
All samples seem to be positive!!
Inoculum of I62, I63, I64, I65 and I66 of 3 colonies in LB+Amp 5ml.
September, 24th
September, 25th
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