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Latest revision as of 06:48, 26 September 2007

Information obtained from:

Roche Applied Science

We used four enzymes for the standard assembly:

• EcoRI

Protocol obtained from: EcoRI product information

Pdf link: Pdf

• XbaI

Protocol obtained from: XbaI product information

Pdf link: Pdf

• SpeI

Protocol obtained from: SpeI product information

Pdf link: Pdf

• PstI

Protocol obtained from: PstI product information

Pdf link: Pdf

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Standard Assembly

Standard Assembly is done in such way that the insert is intended to be larger than 500 pb. This means that if we are to ligate a promoter with a coding sequence, the promoter BioBrick will be the 'vector' and the coding sequence BioBrick will be the 'insert'.

Usually we digest following the recommendations from the commercial information:

• BioBrick as vector (i.e., the part that, apart from the BioBrick, bears the origin of replication after the ligation)

DNA	1 ug
enzyme	1 uL
enzyme	1 uL
H buffer	2 uL
ddwater	up to 20 uL

→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC

• BioBrick as insert (i.e., part that only has the BioBrick)

DNA	1 ug
enzyme	1uL
enzyme	1 uL
H buffer	2 uL
ddwater	up to 20 uL

→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC

Changing the plasmid backbone

If we want to change the backbone (in order to change the antibiotic resistance) of a given plasmid, substituting the original BioBrick, for the new one, we will use EcoRI and PstI, instead of doing a Standard Assembly.

We follow this recipe:

• vector

DNA	1 ug
enzyme	1 uL
enzyme	1 uL
H buffer	2 uL
ddwater	up to 20 uL

→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC

• insert

DNA	1 ug
enzyme	1uL
enzyme	1 uL
H buffer	2 uL
ddwater	up to 20 uL

→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC