HpaII R6311

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	Directional PCR and sequencing were used to confirm the correct orientation and sequences of chemokine gene inserts. P6-secretion signal-chemokine gene cassettes were then excised from pBR322 by ClaI restriction and inserted into pGK12, which was restricted with HpaII (Promega).

Protocol tips
Directional PCR and sequencing were used to confirm the correct orientation and sequences of chemokine gene inserts. P6-secretion signal-chemokine gene cassettes were then excised from pBR322 by ClaI restriction and inserted into pGK12, which was restricted with HpaII (Promega).

Publication protocol

Transducing-particle suspensions (0.2 ml) prepared as described above were subjected to phenol-chloroform extraction and ethanol precipitation as described previously (25). The resultant DNA was HpaII restricted for 8 h, and fragments were separated on a 0.3% agarose gel. DNA was blotted onto a Hybond-N+ nylon membrane (GEC Healthcare) as described previously (25) and probed with digoxigenin (DIG)-labeled linearized pGK12 by using DIG high-prime DNA labeling and detection starter kit II per the instructions of the manufacturer (Roche, Germany).

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