IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/06/14

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Standardization

Today I went with Miguel A. Ramírez to decide which is the best way to standardize the pBBRMCS-5 plasmid, we noticed that the plasmid has an insertion site with many restriction sites:

 KpnI
 ApaI
 Site W
 Site X
 Site y
 Site Z
 HindI
 EcoR1
 PstI
 SpeI
 XbaI
 B
 SacI

So we want to eliminate the most of the restriction sites and obviously we need to eliminate the prefix and suffix sites (italic). To do it we are going to cut in the ApaI and SacI site, on the other hand we're going to amplify a region which contains the suffix, an RFP and the prefix of the plasmid pSB1T3, this PCR amplification will be done with primers containing ApaI and SacI respectively, thereafter we'll can insert this amplification in our pBBRMCS-5.

The primers design will be done by Melisa Rivas

Plasmid Isolation

We need both pBBRMCS-5 and pSB1T3, so today I isolate them from E. coli. The isolation was made following this

I did 5 isolations:

1. pBBRMCS-5, this was made with 30 microliters of TE-RNAse.
2. pBBRMCS-5, this was made with 50 microliters of TE-RNAse.
3. pBBRMCS-5, this was made with 50 microliters of TE-RNAse and 5 mililiters of liquid medium.
4. pSB1T3, this was made with 50 microliters of TE-RNAse
5. pSB1T3, this was made with 50 microliters of TE-RNAse and 5 mililiters id liquid medium

After I did the corresponding electroforesis gel(120V, 35 min):

Lanes 1. Ladder 2. Isolation 1 3. Isolation 2 4. Isolation 3 5. Isolation 4 6. Isolation 5 7. Ladder

gel

I don't know if the 2nd an 3rd lane show a band at the top so I did the gel again


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