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| Line 32: |
Line 32: |
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| | ==August, 23rd== | | ==August, 23rd== |
| - | Resuspension from iGEM 2010 Distribution Kit of <partinfo>BBa_J13002</partinfo> (Plate 1, Well 13B) and <partinfo>BBa_I13522</partinfo> (Plate 2, Well 8A).
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| - |
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| - | 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.
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| - |
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| - | Transformation of 15-4C5 (1 ul) into in re-competentized ''E. coli'' TOP10 carrying <partinfo>BBa_T9002</partinfo>.
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| - |
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| - | 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.
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| | ==August, 24th== | | ==August, 24th== |
| - | All plates showed colonies.
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| - |
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| - | 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.
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| - |
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| - | 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.
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| - |
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| - | ----
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| - |
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| - | Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:
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| - |
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| - | {| border='1' align='center'
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| - | |I7-A || I8-5 D|| I9-1
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| - | |-
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| - | |I10-B|| I12-2|| A2
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| - | |-
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| - | |<partinfo>BBa_B0032</partinfo>|| I144C5-T9002|| I164C5-T9002
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| - | |-
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| - | |I174C5-T9002|| I184C5-T9002|| I194C5-T9002
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| - | |-
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| - | |ENTERO4C5-T9002|| ||
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| - | |}
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| - |
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| - | Glycerol stock was prepared for ENTERO4C5-T9002.
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| | ==August, 25th== | | ==August, 25th== |
| - |
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| - | Cultures were diluted (5ul in 2ml LB+antibiotic) for TECAN test:
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| - |
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| - | {| border='1' align='center'
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| - | |I7-A || I8-5 D|| I9-1
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| - | |-
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| - | |I10-B|| I12-2|| A2
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| - | |-
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| - | |<partinfo>BBa_B0032</partinfo>|| I144C5-T9002|| I154C5-T9002
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| - | |-
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| - | |I164C5-T9002|| I174C5-T9002|| I184C5-T9002
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| - | |-
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| - | |I194C5-T9002|| ENTERO4C5-T9002||
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| - | |}
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| - |
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| - | Glycerol stock was prepared for I154C5-T9002.
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| - |
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| - | ----
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| - | The following cultures were miniprepped and quantified with Nanodrop:
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| - | *I26: 67,5 ng/ul
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| - | *<partinfo>BBa_K105012</partinfo>: 60,9 ng/ul
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| - | *<partinfo>BBa_B0034</partinfo>: 64,5 ng/ul
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| - | *<partinfo>BBa_F2620</partinfo>: 91,8 ng/ul
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| - | *I20M-1: 191,8 ng/ul
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| - | *I20A-1: 176,2 ng/ul
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| - | *I21M-3: 156,2 ng/ul
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| - | *I21-4: 76,3 ng/ul
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| - | *I0: 121,2 ng/ul
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| - | *INTEIN: 143,9 ng/ul
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| - | *<partinfo>J13002</partinfo>: 31,9 ng/ul
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| - |
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| - | and than digested as follows for four hours:
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| - | {| border="1" align='center'
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| - | | ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1'' || ''Enzyme 2'' || ''Buffer H''
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| - | |-
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| - | | I26 || Insert || 25 || 21,5 || 0 || 0,5 EcoRI || 0,5 SpeI || 2,5
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| - | |-
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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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| - | |-
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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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| - | |-
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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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| - | |-
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| - | | I20M-1 || Insert/Screening || 25 || 8 || 13,5 || 0,5 E || 0,5 S || 2,5
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| - | |-
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| - | | I20M-1 || Insert/Screening || 25 || 8 || 13,5 || 0,5 XbaI || 0,5 PstI || 2,5
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| - | |-
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| - | | I20A-1 || Insert/Screening || 25 || 13 || 8,5 || 0,5 E || 0,5 S || 2,5
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| - | |-
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| - | | I20A-1 || Insert/Screening || 25 || 13 || 8,5 || 0,5 X || 0,5 P || 2,5
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| - | |-
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| - | | I21-4 || Insert/Screening || 25 || 13 || 8,5 || 0,5 E || 0,5 S || 2,5
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| - | |-
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| - | | I21-4 || Vector || 25 || 13,1 || 8,4 || 0,5 E || 0,5 X || 2,5
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| - | |-
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| - | | I0 || Vector || 25 || 8,2 || 13,3 || 0,5 E || 0,5 XbaI || 2,5
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| - | |-
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| - | | INTEIN || Insert || 25 || 10,5 || 11 || 0,5 E || 0,5 S || 2,5
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| - | |-
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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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| - | |-
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| - | |}
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| - | Two medium gel were prepared and samples were loaded and gel run/cut.
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| - |
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| - | {|align="center"
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| - | |[[Image:UNIPV10_18_8_10_vettori_fasine.jpg|thumb|300px|center|Vectors digested and gel run/cut]] || [[Image:UNIPV10_18_08_10insertifasine.jpg|thumb|300px|center|Inserts digested and gel run/cut]]
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| - | |}
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| - |
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| - | 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.
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| - |
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| - | Gel extraction gave these results:
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| - | *I0 (E-X): 20,8 ng/ul
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| - | *<partinfo>BB_F2620</partinfo> (E-S): 13,5 ng/ul
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| - | *INTEIN (E-S): 6,5 ng/ul
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| - | *<partinfo>BB_B0034</partinfo> (E-X): 23,2 ng/ul
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| - | *I20M-1 (E-S): 7,4 ng/ul
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| - | *<partinfo>BB_J13002</partinfo> (S-P): 12,5 ng/ul
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| - | *I21-4 (E-S): 7,2 ng/ul
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| - | *I21-4 (E-X): 24,7 ng/ul
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| - | *<partinfo>BBa_K105012</partinfo> (E-X): 23,1 ng/ul
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| - | *I26 (E-S): 3,4 ng/ul
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| - | *I20M-1 (X-P): 6,5 ng/ul
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| - | They were stored ON at -20°C.
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| | | | |
| | ==August, 26th== | | ==August, 26th== |
| - | Extra E-X digestion (four hours) of previously miniprepped INTEIN
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| - | {|align="center" border="1"
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| - | |'''Culture'''||'''Kind'''||'''H2O'''||'''DNA'''||'''Enzyme1'''||'''Enzyme2'''||'''Buffer H'''
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| - | |-
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| - | |INTEIN || Vector || 13,5 ul||7 ul|| 1 ul EcoRI||1 ul XbaI||2,5 ul
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| - | |-
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| - | |}
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| - | Sample was loaded into a small agarose gel, run and cut.
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| - |
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| - | After gel estraction was quantified with Nanodrop: 19,6 ng/ul.
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| - |
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| - | Finally were performed the following ligations:
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| - | *I31: I20M-1 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
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| - | *I32: I20M-1 (E-S) + I21-4 (E-X)
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| - | *I33: I20M-1 (E-S) + I0 (E-X)
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| - | *I34: I21-4 (E-S) + I0 (E-X)
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| - | *I35: I21-4 (E-S) + <partinfo>BBa_K105012</partinfo> (E-X)
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| - | *I36: I21-4 (E-S) + I21-4 (E-X)
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| - | *I37: INTEIN (E-S) + I0 (E-X)
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| - | *I38: <partinfo>BBa_F2620</partinfo> + <partinfo>BBa_B0034</partinfo>
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| - | *I39: <partinfo>BBa_J13002</partinfo> + I20M-1 (X-P)
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| - | *I40: I26 (E-S) + INTEIN (E-X)
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| - | *I41: I26 (E-S) + I0 (E-X)
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| | | | |
| | ==August, 27th== | | ==August, 27th== |
| - | 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.
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| - |
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| - | ----
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| | | | |
| | ==August, 28st== | | ==August, 28st== |
| - | Plates with ligations I31..I41 were stored at +4°C. Colonies will be screened ASAP.
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| | | | |
| | ==August, 29nd== | | ==August, 29nd== |
"
