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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 it.

• We have achieved a nano spectrophotometer and readings are 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 miniprep method

we have a low concentration of vector plasmid.

We have to work an try to get more plasmid and used diluted PCR products

is not possible achieve ligations with this vector an insert ratio.

Our advisor proposed use low Melting agarose

to extract the plasmid from the gel.

Tuesday

We ran with a low meelting agarose and cut off the band

we precipitated using alchol and salts in order

to get enough vector to do the ligations.

Wenesday

Plan

• We set up all to do the ligations.
• We digested both vector and insert with EcoRI and PstI .
• We ran a low melting agarose gel 1 hour at 80 volts, then cut off the band from gel.

At Claudia's lab we tried three different methods to recover DNA from gel.

• '

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