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


Week #4

27th September - 01 October 2010


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.


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.




  • 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



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


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!!!!

Labo 254.JPG