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		+	'''We used the follow notation.'''
		+	*'''For vector purified from band:              Pu'''
		+	*'''For iGEM's vector (Vial with green cover):  P'''
		+	*'''Vector  only digest:                        S'''

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Revision as of 05:09, 25 October 2010

Contents 1 Week #4 2 27th September - 01 October 2010 2.1 Monday 2.1.1 Well we are stuck with the vector this week we have to get vector is our main objetive. 2.2 Tuesday 2.2.1 We run with a low meelting's agarose prove to extract band tomorrow 2.2.2 we do a protocol to precipit with alchol an salts our objetive is do it all 2.2.3 to get vector and do the ligations. 2.3 Wensday 2.3.1 Plan 2.4 Thursday 2.4.1 Plan

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 mini prep

we have a low concentration of vector’s plasmid DNA.

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

is not posible to do ligations with this ratio betwen 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 going to cut with EcoRI and PstI both vector and insert.
• We'll run a low melting gel slow 1 hour and an half 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 a dilutions and do the ligation

For this we do 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