User:Ferdinando Pucci/Notebook/Cloning/2008/05/10

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Tie2-GFP (#416) and tetO7-GFP(no Xho1) (#969) in CCL transfer plasmid
Yesterday ON digestion with: Gel loading order:
 * NarI+AgeI for #969 and #277 (PGK-GFP)
 * NarI for #416 and #277

Some undigested is present for #969 and maybe #416 but it should not interfer with subsequent steps. It could be due to the thermal cycler lid not heated.

I purify the #416 and #227-NarI for the second digestion. Using the Qiagen miniprep kit:
 * 5X of PB buffer volume
 * 13.000 RPM 1'
 * Wash with 750ul of PE buffer
 * 13.000RPM 1'
 * Empty and centrifuge again
 * 40ul of water at 37°

Add 5ul of Neb3+BSA and 5ul of SalI. 37° 3h.

Gel loading order:

Actually #416 is partially undigested. 277 Nar+Sai is ok.

Bands to cut

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