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:

  • NarI+AgeI for #969 and #277 (PGK-GFP)
  • NarI for #416 and #277

Gel loading order:

1Kb 277 Nar p277 277 Nar+Age p416 416 Nar p969 969 Nar 1Kb

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:

1Kb 416 Nar+Sal p416 277 Nar+Sal p277 1Kb

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

Bands to cut

Plasmid Band Loading order
277 5Kb (Sal1+Nar1) 4
277 5.7Kb (Age1+Nar1) 3
416 6Kb (Sal1+Nar1) 2
969 2Kb (Age1+Nar1) 1
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