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

From 2010.igem.org

(Difference between revisions)
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*'''We prepared all to do ligations PsB1C3 with our Pcr's.'''
*'''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.'''
*'''For this we going to cut with EcoRI and PstI both vector and insert.'''
-
*'''We'll run a low melting gel slow 2 hours then purified from the band.'''
+
*'''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 gelof which two of'''
+
'''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.'''
'''them where purified by Axigen kit the third one by a diferent kit.'''
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{| border="1"
{| border="1"
-
 
|-
|-
|DNA
|DNA
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==''Thursday''==
==''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.'''
 +
 +
{| border="1"
 +
|-
 +
|Insert [20ng/μl]
 +
|5μl
 +
|-
 +
|Vector
 +
|1μl
 +
|-
 +
|Reaction buffer
 +
|1μl
 +
|-
 +
|T4 DNA Ligase
 +
|1μl
 +
|-
 +
|H2O
 +
|2μl
 +
|-
 +
|Total
 +
|10μl
 +
|}

Revision as of 05:00, 25 October 2010


Contents

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.

Imagen5.gif


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
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