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From 2010.igem.org

Week #4

27th September - 01 October 2010

Monday

Well...we are stuck with the vector this week we have to get vector is our main objetive.

• We have achived a nanodrop readings as below

	ng/μl	260/280	260/230
PsB1C3	0.60	-0.73	-0.0
PCR 1 red	427.70	1.71	1.71
PCR 2 red	483.20	1.74	1.71
PCR 3 red	410.70	1.76	2.02
PCR 4 red	431.05	1.61	1.88
PCR 1 blue	533.35	1.71	1.75
PCR 2 blue	536.00	1.72	1.82
PCR 3 blue	577.50	1.69	1.59
PCR 4 blue	627.55	1.70	1.56
PsB1C3	4.10	2.62	1.01

• Yep our trouble is the lisis alcaline method to do the miniprep

we have a low concentration of vector’s plasmid.

We have to work an try to get more plasmid and make PCR's dilutions

is not posible to do ligations with this ratio between vector an insert.

On this step we advisor has proposed a method via Low Meelting agarose

to geting out plasmid from the band.

Tuesday

We run with a low meelting's agarose prove to extract band tomorrow

we do a protocol to precipit with alchol an salts our objetive is do it all

to get vector and do the ligations.

Wensday

Plan

• We prepared all to do ligations PsB1C3 with our Pcr's.
• For this we made a digest with EcoRI and PstI both vector and insert.
• We have run a low melting gel slow 1 hour at 80 volts, then purified from the band.

At Claudia's lab we cut tree bands from 2% agarose's gel of which two of

them where purified by Axigen kit the third one by a diferent kit.

The digestion was done with following amounts.

DNA	20μl
Buffer NB2	3μl
EcoRI	2μl
PstI	2μl
H2O	2.4μl
BSA	0.6μl
Total	30μl

Thursday

Plan

We going to proceed to do dilutions then ligations

For this we did the following accounts

Poner las fórmulas usadas en un formato bonito

We did the ligations using the following amounts.

Insert [20ng/μl]	5μl
Vector	1μl
Reaction buffer	1μl
T4 DNA Ligase	1μl
H2O	2μl
Total	10μl

We used the follow notation.

• For vector purified from band: Pu
• For iGEM's vector (Vial with green cover): P
• Vector only digest: S

After one hour of ligation (time recomended by the protocol) we transformed by thermic shock

and left overnight plates to 37º

Friday

Today we discused what we going to do with the AFP (anti freeze protein),

We going to transform by thermic shock plating on kanamicina medium, then prepare

miniprep and cut with Xba and PstI we have to ligate at J13002 this as blackbone

the blackbone will cut with SpeI and PstI.

Meanwhile our results of previous day ligations.

• PCR1 Lig S Positive
• PCR2 Lig S Positive
• PCR3 Lig S Positive
• PCR4 Lig Pu Positive
• PCR4 Lig S Positive