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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.

Wednesday

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 two different kits to recover DNA from gel.

• Mobio ultra clean Microbial DNA kit
• Axygen.....

The digestions were done with following amounts.

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

Thursday

Plan

We then proceed to diluted PCR fragments

For this we did the following operations:

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 denomination.

• Band purified vector : Pu
• iGEM's vector (Vial with green cover): P
• Digested vector: S

After one hour ligation we transformed by heat shock

and kept overnight the plates at 37ºC

Friday

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

We decided transform by heat shock and plating it on kanamicina medium, then prepare

miniprep plasmid and cut it with XbaI and PstI. We have to ligate it to the backbone J13002

the backbone had to be cut with SpeI and PstI.

Meanwhile, here are the previous day ligations.

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