Team:UNIPV-Pavia/Calendar/June/settimana4

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Revision as of 09:04, 5 July 2010

JUNE: WEEK 4

June, 21st

Planning of the activity of the week. Freezer cleaning: for each ligation, we choosed the correct clone and stored it in the iGEM 2010 ligations box. All these cloned were gel screened and sequenced and re correct! The colonies we choosed are:

colony choosed	ligation name
I0-2	I0
I1-2	I1
I2-1	I2
I3-1	I3
I4-2	I4
I5-1	I5
I6-2	I6

Other colonies resulted psitives at the screening but were NOT sequenced and are stored in the iGEM 2010 cemetery box. These clones are: I1-1, I2-2, I4-1 and I6-3.

June, 22nd

Inoculum of I6, <partinfo>BBa_J23118</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo> from glycerol stock in 5ml LB+Amp. Cultures were grown ON at 37°C 220rpm.

June, 23rd

Cultures incubated ON were all grown. Plasmids were extracted with MiniPrep.

J231xx and I6 digestion

After MiniPrep, purified DNA was quantified with NanoDrop.

I6	193 ng/ul
<partinfo>BBa_J23118</partinfo>	107,9 ng/ul
<partinfo>BBa_J23110</partinfo>	83 ng/ul
<partinfo>BBa_J23114</partinfo>	89,8 ng/ul
<partinfo>BBa_J23116</partinfo>	82,7 ng/ul

Digestion of:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1	Enzyme 2	Buffer H
I6	Insert	25	9,3	11,2	1 XbaI	1 PstI	2,5
I6-bis	Insert	25	9,3	11,2	1 XbaI	1 PstI	2,5
<partinfo>BBa_J23118</partinfo>	Vector	25	9,3	11,2	1 SpeI	1 PstI	2,5
<partinfo>BBa_J23110</partinfo>	Vector	25	12	8,5	1 SpeI	1 PstI	2,5
<partinfo>BBa_J23114</partinfo>	Vector	25	11,2	9,3	1 SpeI	1 PstI	2,5
<partinfo>BBa_J23116</partinfo>	Vector	25	12,1	8,4	1 SpeI	1 PstI	2,5

Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.

Ligations were performed ON at 16°C:

I7: <partinfo>BBa_J23118</partinfo> (S-P)+ I6 (X-P)
I8: <partinfo>BBa_J23110</partinfo> (S-P)+ I6 (X-P)
I9: <partinfo>BBa_J23114</partinfo> (S-P)+ I6 (X-P)
I10: <partinfo>BBa_J23116</partinfo> (S-P)+ I6 (X-P)

NanoDrop quantifications were not reliable, so every ligation was performed with 3ul of vector, 2ul of insert, 3ul of ddH20, 1ul of T4 ligase and 1ul of T4 ligase buffer.

These four promoters from Andersone Promoters Collection were choosed for their strength, measured in Arbitrary Units:

Part	RFP (a.u.)
<partinfo>BBa_J23118</partinfo>	1429
<partinfo>BBa_J23110</partinfo>	844
<partinfo>BBa_J23114</partinfo>	256
<partinfo>BBa_J23116</partinfo>	396

Soon quantitative tests will be performed to quantify each promoter's strength in terms of RPU.

Today we received 2 stabs from registry HQ for <partinfo>BBa_K208001</partinfo> (one for each registry loation of this part),the Silver-fusion compatible BioBrick part that codes for phasin. This part is in a pSB3K3 plasmid. Cultures were straked on LB+Kan (50 ug/ml) agar plates. Plates were incubated ON at 37°C.

June, 24th

June, 25th

Screening of I7, I8, I9, I10

June

week 2

week 3

week 4

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-Soon quantitative testes will be performed to quantify each promoter's strength in terms of RPU.
+Soon quantitative tests will be performed to quantify each promoter's strength in terms of RPU.
+Today we received 2 stabs from registry HQ for <partinfo>BBa_K208001</partinfo> (one for each registry loation of this part),the Silver-fusion compatible BioBrick part that codes for phasin. This part is in a pSB3K3 plasmid. Cultures were straked on LB+Kan (50 ug/ml) agar plates. Plates were incubated ON at 37°C.
 ==June, 24th==