User:Tanguy Chau/Notebook/Research/2008/07/10

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Ligation of R797, R796 into T7 415-1b Vector
Inserts obtained from Tim Lu R797: EcorI-Cecropin-HindIII @ 27.9ng/ul

Apporoximate size/sequence: GAATTC|STOP|AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG|ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC|SWLSKTAKKLENSAKKRISEGIAIAIQGGPR|GGATCCGGCTGCTAACAAAGCCCGAAGCTT
 * ~242bp, with ~660g/mol.bp  ~160,000 g/mol
 * For 0.06pmol of insert, need 9.6 ng of insert
 * Need 0.344 ul of R797 insert

R796: EcoRI-Indolicidin-HindIII @ 15.1ng/ul GAATTC|STOP|AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATG|ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC|ATTCTGCCGTGGAAATGGCCGTGGTGGCCGTGGCGCCGCTGA|GGATCCGGCTGCTAACAAAGCCCGAAGCTT
 * Apporoximate size/sequence:
 * ~253bp, with ~660g/mol.bp  ~160,000 g/mol
 * For 0.06pmol of insert, need 9.6 ng of insert
 * Need 0.635 ul of R797 insert

Ligation Reaction
 * 5 μl	Total volume:
 * 1 ul	insert (0.02–0.06 pmol) or Positive Control Insert or no insert negative control
 * 1 ul	T7Select Vector Arms (0.5 μg; 0.02 pmol)
 * 1.5 ul	10X T4 DNA Ligase Reaction Buffer
 * 10mM ATP
 * 100mMDTT
 * 0.5 ul	Sterile water
 * Hold at 55C for 10 min
 * 1 μl	T4 DNA Ligase (0.4–0.6 Weiss units)
 * Ligate at 16C for 3-16h


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