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AUGUST: WEEK 3
August, 16th
Resuspension from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).
Transformation (1 ul) of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM (Mr.Gene) into 100 ul of E. coli TOP10 home-made competent cells.
Transformation of 15-4C5 (1 ul) into in re-competentized E. coli TOP10 carrying <partinfo>BBa_T9002</partinfo>.
Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.
August, 17th
All plates showed colonies.
Pick of <partinfo>BBa_J13002</partinfo>, <partinfo>BBa_I13522</partinfo>, INTEIN, CREAM, co-trasformed <partinfo>BBa_T9002</partinfo> and inoculum into 1 ml of LB+Amp, LB+Kan, LB+Cm, respectively, to make glycerol stocks. Falcons were than refilled for the minipreps of the following day.
Inoculum of I5, I26, <partinfo>BBa_K105012</partinfo>, <partinfo>BBa_B0034</partinfo>, I20M-1, I20A-1, I21M-3, I21-4, I21A-7, I0 for minipreps of the following day.
Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:
| I7-A | I8-5 D | I9-1
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| I10-B | I12-2 | A2
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| <partinfo>BBa_B0032</partinfo> | I144C5-T9002 | I164C5-T9002
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| I174C5-T9002 | I184C5-T9002 | I194C5-T9002
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| ENTERO4C5-T9002 | |
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Glycerol stock was prepared for ENTERO4C5-T9002.
August, 18th
Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:
| I7-A | I8-5 D | I9-1
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| I10-B | I12-2 | A2
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| <partinfo>BBa_B0032</partinfo> | I144C5-T9002 | I154C5-T9002
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| I164C5-T9002 | I174C5-T9002 | I184C5-T9002
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| I194C5-T9002 | ENTERO4C5-T9002 |
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Glycerol stock was prepared for I154C5-T9002.
The following cultures were miniprepped and quantified with Nanodrop:
- I26: 67,5 ng/ul
- <partinfo>BBa_K105012</partinfo>: 60,9 ng/ul
- <partinfo>BBa_B0034</partinfo>: 64,5 ng/ul
- <partinfo>BBa_F2620</partinfo>: 91,8 ng/ul
- I20M-1: 191,8 ng/ul
- I20A-1: 176,2 ng/ul
- I21M-3: 156,2 ng/ul
- I21-4: 76,3 ng/ul
- I0: 121,2 ng/ul
- INTEIN: 143,9 ng/ul
- <partinfo>J13002</partinfo>: 31,9 ng/ul
and than digested as follows for four hours:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 (ul) | Enzyme 2 (ul) | Buffer H (ul)
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| I26 | Insert | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 SpeI | 2,5
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| <partinfo>BBa_K105012</partinfo> | Vector | 25 | 16,5 | 5 | 0,5 E | 0,5 S | 2,5
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| <partinfo>BB_B0034</partinfo> | Vector | 25 | 15,5 | 6 | 0,5 E | 0,5 S | 2,5
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| <partinfo>BB_F2620</partinfo> | Insert | 25 | 16,5 | 5 | 0,5 E | 0,5 S | 2,5
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| I20M-1 | Insert/Screening | 25 | 8 | 13,5 | 0,5 E | 0,5 S | 2,5
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| I20M-1 | Insert/Screening | 25 | 8 | 13,5 | 0,5 XbaI | 0,5 PstI | 2,5
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| I20A-1 | Insert/Screening | 25 | 13 | 8,5 | 0,5 E | 0,5 S | 2,5
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| I20A-1 | Insert/Screening | 25 | 13 | 8,5 | 0,5 X | 0,5 P | 2,5
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| I21-4 | Insert/Screening | 25 | 13 | 8,5 | 0,5 E | 0,5 S | 2,5
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| I21-4 | Vector | 25 | 13,1 | 8,4 | 0,5 E | 0,5 X | 2,5
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| I0 | Vector | 25 | 8,2 | 13,3 | 0,5 E | 0,5 XbaI | 2,5
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| INTEIN | Insert | 25 | 10,5 | 11 | 0,5 E | 0,5 S | 2,5
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| <partinfo>BB_J13002</partinfo> | Vector | 25 | 21,5 | 0 | 0,5 S | 0,5 P | 2,5
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Two medium gel were prepared and samples were loaded and gel run/cut.
 Vectors digested and gel run/cut |  Inserts digested and gel run/cut |
All samples were correctly digested and run; both I20M-1 and I20A-1 are positive, so we decided to use I20M-1; this new screening for I21-4 showed again that it's correct.
Gel extraction gave these results:
- I0 (E-X): 20,8 ng/ul
- <partinfo>BB_F2620</partinfo> (E-S): 13,5 ng/ul
- INTEIN (E-S): 6,5 ng/ul
- <partinfo>BB_B0034</partinfo> (E-X): 23,2 ng/ul
- I20M-1 (E-S): 7,4 ng/ul
- <partinfo>BB_J13002</partinfo> (S-P): 12,5 ng/ul
- I21-4 (E-S): 7,2 ng/ul
- I21-4 (E-X): 24,7 ng/ul
- <partinfo>BBa_K105012</partinfo> (E-X): 23,1 ng/ul
- I26 (E-S): 3,4 ng/ul
- I20M-1 (X-P): 6,5 ng/ul
They were stored ON at -20°C.
August, 19th
Extra E-X digestion (four hours) of previously miniprepped INTEIN
| Culture | Kind | H2O | DNA | Enzyme1 | Enzyme2 | Buffer H
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| INTEIN | Vector | 13,5 ul | 7 ul | 1 ul EcoRI | 1 ul XbaI | 2,5 ul
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Sample was loaded into a small agarose gel, run and cut.
After gel estraction was quantified with Nanodrop: 19,6 ng/ul.
Finally were performed the following ligations:
- I31: I20M-1 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
- I32: I20M-1 (E-S) + I21-4 (E-X)
- I33: I20M-1 (E-S) + I0 (E-X)
- I34: I21-4 (E-S) + I0 (E-X)
- I35: I21-4 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
- I36: I21-4 (E-S) + I21-4 (E-X)
- I37: INTEIN (E-S) + I0 (E-X)
- I38: <partinfo>BBa_F2620</partinfo> + <partinfo>BBa_B0034</partinfo>
- I39: <partinfo>BBa_J13002</partinfo> + I20M-1 (X-P)
- I40: I26 (E-S) + INTEIN (E-X)
- I41: I26 (E-S) + I0 (E-X)
August, 20th
Ligations of previous day were transformed (1 ul) into 100 ul of home-made competent cells 'E. coli DH5-alpha. They were than plated on LB+Amp agar plates and incubated ON at 37°C.
Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).
 Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.
Tecan Test
August, 21st
Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.
Glycerol stocks and miniprep for MyCrim 1:9.
Quantifications:
| Culture | Quantification (ng/ul)
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| MyCRIM 1 | 59
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| MyCRIM 2 | 145
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| MyCRIM 3 | 108
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| MyCRIM 4 | 125
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| MyCRIM 5 | 65
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| MyCRIM 6 | 72
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| MyCRIM 7 | 97
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| MyCRIM 8 | 127
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| MyCRIM 9 | 151
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Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:
| DNA | Buffer B | H2O | EcoRI | HindIII
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| 2 ul | 2.5 ul | 18.5 ul | 1 ul | 1 ul
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 Screening digestions: MyCRIM (from 1 to 9).
The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.
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