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Team:Mexico-UNAM-CINVESTAV/Notebook/Week Four
From 2010.igem.org
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==='''Well...we are stuck with the vector. This week we have to get it.'''=== | ==='''Well...we are stuck with the vector. This week we have to get it.'''=== | ||
| - | *'''We have | + | *'''We have achieved a nano spectrophotometer and readings are below:''' |
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==='''Plan'''=== | ==='''Plan'''=== | ||
| - | *'''We | + | *'''We set up all to do the ligations.''' |
| - | *''' | + | *'''We digested both vector and insert with ''EcoR''I and ''Pst''I .''' |
| - | *'''We | + | *'''We ran a low melting agarose gel 1 hour at 80 volts, then cut off the band from gel.''' |
| - | '''At Claudia's lab we | + | '''At Claudia's lab we tried three different methods to recover DNA from gel.''' |
| - | ''' | + | *'''''' |
'''The digestion was done with following amounts.''' | '''The digestion was done with following amounts.''' | ||
Revision as of 21:41, 26 October 2010
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
