View source for Submitting 10 casein and FAM20C in pD1214 plasmids for sequencing 30Sep2014

'''September 30, 2014: DNA pick-up by [http://sequetech.com/ Sequetech] to sequence 16 samples, detailed below'''
*'''Location:''' BioCurious

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

*'''Aims:''' Confirm correct DNA sequence of our casein and FAM20C inserts in pD1214 vector. Trouble shoot Electra cloning negative control positive colonies (See [[Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014]], "Results and Discussion" September 28, 2014.) Confirm identity of unclearly labeled DNA, prob. hKcasein Kex+ in pD1214.

*'''Sequencing Strategy & Methods:'''
**Submitting 10uL aliquots of 16 samples, detailed in table below, including 10 new casein and FAM20C in pD1214 constructs.
***4 samples submitted in duplicate -- plasmid preps performed on two separate ''E. coli'' clones: 
**** P.FAKS.humBetaS:pD1214, as previously problematic construct with few transformed colonies (See [[Troubleshooting failed 12Sep2014 transformation of P.FAKS.humBeta.S]] for details.)
**** P.FAKSbovBetaB:pD121, FAM20C Kex(+):pD1214 and FAM20C Kex(-):pD1214, as plasmid inserts may not be what we expect. (See [[Cloning bovine beta casein, Fam20C (Kex + & -), from 27Sep2014]], "Results and Discussion" September 28, 2014.)
***NOTE: DNA concentration for bovAlphaS2 Kex(+) very low. May have to repeat plasmid prep.
**''' ''Sequetech rep. collected samples at ~2:00pm today.'' '''
**All except hkk+R to be sequenced in two reactions: the forward (with primer ''TEF-264-FW'') and reverse (with primer ''PMYR3'') directions. Sample hkk+R sampled in single direction as was likely the sample already sequenced in both directions, September 5, 2014.
***''TEF-264-FW'' is a custom primer synthesized through Sequetech: primer sequence recommended by DNA 2.0, suppliers of pD1214. This primer will be maintained in-house for future sequencing reactions. (''Note change from sequencing described August 6, 2014, as previous forward primer,'' Alpha-factor 146-forward, '' anneals to the FAKS sequence, now inside our redesigned DNA inserts.'') See for primer map.
***''PMYR3'' is an in-house Sequetech primer.

 '''Primer Sequences''' 
 ''TEF-264-FW'': 5’ TCGATGACCTCCCATTGA 3’
  Binds to TEF promoter and reads through in the 5'-3' direction (N-terminus of signal peptide).
  
 ''PMYR3'': 5' CTTCCTTTTCGGTTAGAG 3' 
  Binds to the terminator (CYC1) and reads through in the 3'-5' direction (C-terminus of casein).

'''Location of sequencing primers indicated by small purple arrows on map of pD1214:'''
[[File:PD1214 Map.png]]

'''Details for 31 sequencing reactions on 16 samples:'''

[[File:Samples details from Sequetech sequence pick-up, 30Sep2014.png|600px]]

'''Sequencing Analysis Summary Table (October 1, 2014)'''

[[File:Screen_Shot_2014-10-01_at_4.42.46_PM.png|600px]]

''' ''Clone 9A is also poor. Need to remove from stocks.'' '''

*'''Discussion:''' Of the 11 sequencing reactions we were able to evaluate (excluding all FAM20C, as insufficient length, and Electra cloning negative, as didn't align), there were confirmed issues with 3/11 (27.3%): incorrect sequences for bovine alpha S2 Kex+ (#3) & human beta (#4.2) and what looks like poor template DNA for human kappa Kex- (#5). Given that rate, suggest prepping and submitting two separate clones for each construct in the future.