By Sandra Boynton
Soon after darkish, upon the seashore, we sing a hippo music, and if you’re feeling within the temper, we are hoping you’ll sing alongside: “Belly stomach Button, you’re oh so fantastic. Ooo, stomach Button, I’m so satisfied you’re mine.”
glossy and durable, and that includes an outstanding (navel-shaped, evidently) die-cut conceal, the Belly Button booklet presents enduring, giggly, read-aloud fun.
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Manno CS et al (2006) Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12(3):342–347 19. Testa F et al (2013) Three-year follow-up after unilateral subretinal delivery of adenoassociated virus in patients with Leber congenital Amaurosis type 2. Ophthalmology 120(6): 1283–1291 20. Inagaki K et al (2007) The role of DNA-PKcs and artemis in opening viral DNA hairpin termini in various tissues in mice. J Virol 81(20):11304–11321 21.
Add 2 μl of dH2O or 10 mM Tris-HCl solution as a no template control. 12. Seal the plate and spin down @ 1800 rpm for 10 s. 13. Perform the Q-PCR in a thermocycler using the manufacturer’s recommended conditions specific to the primer annealing temperature. 7 Southern Dot Blot for Vector Genome: Sample Preparation 1. In an appropriate tube or well add 10 μl of the CsCl vector fraction to 100 μl of DNaseI digestion buffer (solution 1). For final titer determination by this method a twofold dilution series can be used (in triplicate) starting with a max of 10 μl of the dialyzed vector preparation.
10. Run the gel at 15 V overnight in alkaline gel electrophoresis buffer (see Note 20). 11. When gel has finished running cut the top wells off and any other part of the gel that may not be needed. 12. Soak the gel in transfer buffer for 10 min with mild agitation. 13. Measure the size of the gel, so you can cut your filter paper and HyBond-XL membrane accordingly. 14. Perform Southern transfer using standard procedures [39, 40]. 15. Once the transfer is complete UV cross-link the membrane. 16.