SubmittingFam20COct

'''October 14, 2014: DNA pick-up by [http://sequetech.com/ Sequetech] to sequence 2 samples, detailed below'''
*'''Location:''' BioCurious

*'''Participants:''' Rachel (set up sample pick-up), Emi (gave samples to Sequetech rep)

*'''Notes:''' Rachel

*'''Aims:''' The Fam20C Kex + & Kex - inserts are too long to obtain complete reads with primers that anneal externally to the insert (see [[Submitting 10 casein and FAM20C in pD1214 plasmids for sequencing 30Sep2014]].) To complete sequencing coverage, we are resubmitting these two constructs for sequencing with newly designed primers that anneal within the insert region. 

*'''Sequencing Strategy & Methods:'''
**Details about samples submitted for sequencing in table, below.
**Submitting 10uL aliquots of 2 samples: Sap.FAKS.hFam20C(Kex+).Sap:pD1214 and Sap.Faks.hFam20C(Kex-).Sap:pD1214 from [[Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014]]. Submitting clones 9A and 10A. ''' ''Should have been 9B; 9A is "bad" per CR.'' '''
**''' ''Sequetech rep. collected samples at ~2:00pm today.'' '''
**All to be sequenced in '''three''' reactions: two forward primers (''Fam20C For 640'' & ''Fam20C For 1035'') and one reverse (''FAM20C Rev 1205''). All primers designed 09Oct2014 by RVC project members.

 '''Primer Sequences''' 
    
 ''Fam20C For 640'': 5' - CCTGCTTAGAGATCCA - 3' 
  Binds internally in Sap.Faks.hFam20C(Kex + & Kex-).Sap gBlock DNA and reads through in the 5'-3' direction.
 
 ''Fam20C For 1035'': 5' - ATTGATTATGACCTTCCA -3'
  Binds internally in Sap.Faks.hFam20C(Kex + & Kex-).Sap gBlock DNA downstream of ''Fam20C For 640'' and reads     
  through in the 5'-3' direction.
 
 ''FAM20C Rev 1205'' : 5'-ATTCTACCAGCAACAG -3'
  Binds internally in Sap.Faks.hFam20C(Kex + & Kex-).Sap gBlock DNA and reads through in the 3'-5' direction.
  

'''Details for 6 sequencing reactions on plasmid DNA from 2 clones:'''

[[File:Sample_details_from_Sequetech_sequence_pick-up%2C_14Oct2014.png]]

=October 15, 2014=
'''Sample 10A sequenced perfectly. Read to be transformed into yeast!'''
*Need to submit FAM20 Kex + clone 9B for this full-coverage sequencing.