Team:Mexico-UNAM-CINVESTAV/Notebook/Week Four

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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.

Imagen5.gif


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

Labo 254.JPG