8/22/14

Molecular  Entomology  
Laboratory,  Fall  2014  

Tue  and  Thr  

1:30  to  3:30PM  

Goals  
Students  who  do  not  have  experience  in  Molecular  Biology  will  be  exposed  
to  molecular  techniques  frequently  used  in  Entomological  science.    
 
Learning  the  strengths  and  piLalls  of  molecular  techniques  and  uMlize  them  
in  the  students’  research.    !!!You  can  bring  your  own  sample!!!  
 
Learning  who  to  talk  with  for  designing  experiments  and  trouble  shooMng.  
 
•  Laboratory  safety  and  basic  concepts  of  bench  work  (Park)  
•  IsolaMon  of  DNA  and  polymerase  chain  reacMon  (Park)  
•  Analysis  of  nucleic  acids  (Zurek/Ghosh)  
•  Molecular  populaMon  geneMcs  and  evoluMon  (Ragland)  
•  Working  with  RNA  (OrMgao)  
•  RNA  interference  (Zhu)  
•  Analysis  of  protein  (Marshall)  

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Schedule  

Instructor:  Yoonseong  Park  ypark@ksu.edu  

A  volunteering  helper:  Krissana  Ruang-­‐rit  <krissanar@ksu.edu>  

Grade  in  Park’s  porMon  of  the  class  

A  report  combining  everything  done  in  the  laboratory  

Genomic  DNA  prep.  
DNA  quanMficaMon  
Agarose  gel  electrophoresis  
PCR  
Sequencing  

Manuscript  style,  single-­‐spaced,  12  font,  no  page  limit,  but  

preferred  to  be  less  than  3  pages  with  less  than  5  separate  
figures.  

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Safety  

If  you  have  not  done,  read  the  “Safety  pracMces”.  

Highlights  of  the  Safety  in  this  Class  

General  safety  rules  are  all  applied.  

 Read  safety  pracMces  
 Safety  training  you  had  from  your  lab  coordinator  is  equally  applied  to  this  class  
 
We  will  avoid  to  use  hazardous  chemicals/reagents  in  the  class  (Park  secMon).  
 Do  not  drink  and  eat  in  the  class.  
 Wear  gloves  (protecMng  sample  from  your  fingerMp…)  
   
   
The  most  frequent  safety  issues  in  molecular  lab  are  
 -­‐Organic  solvent  (phenol,  chloroform)  
 -­‐EtBr  
 -­‐Formaldehyde  
 
Each  instructor  will  provide  specific  instrucMons  for  safety  in  each  secMon.  

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How  to  avoid  contaminaMon?    

Wear  gloves  
 
Change  pipeBe  Cps  for  different  samples  (different  chemicals,  DNA,  RNA,  
concentraMons)  
 
Choice  of  water  
 
•  Autoclave  does  help  only  for  killing  bacteria  (but  not  chemical  decontaminaMon  
including  DNA)  
•  Other  way  to  prepare  bacteria-­‐free  water  is  0.22mm  filtered  water  
•  Fresh  18GΩ  water  (disMlled-­‐deionized  water,  ddH2O)  is  the  best    
 
Separate  storages  of  different  types  of  sample  shelves  or  freezer  
 
Separate  pipeBe  sets  for  different  purpose  

How  to  pipele  

 

Images  from  Wikipedia  

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How  to  pipele  

 
-­‐Choose  the  size  of  the  pipele  (P10,  P100,  P1000)  
 
1.  Press  the  bulon  to  the  first  stop.  
2.  Dip  the  top  into  the  soluMon.  
3.  Gently  release  the  bulon  (aspiraMng)  
 Depending  on  the  soluMon  type  this  step  need  to  be  carefully  done  
4.    Dispense  the  liquid  to  receiving  tube  by  pressing  the  bulon  
to  all  the  way  to  second  stop.  
   
-­‐Tricky  techniques  
•  Mixing  soluMon  
•  Viscosity  of  the  soluMon  (Aqueous,  Viscous,  volaMle)  
•  RepeMMve  pipenng  to  several  different  tubes  

MulMchannel  pipele  
Other    frequently  used  pipeles  
 

Lager  volume   Electronic  pipele  

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Metric  prefixes  frequently  used.  

Basic  of  molecular  bench  work  

 
CalculaMon  of  concentraMon  and  absolute  amount.  
•  0.01%  (wt/v)  
•  1:100  
•  1  μmoles  
•  1  μmoles/L  
•  1  μM  
•  1  ppm  
1  moles  is  6.02  x  1023  molecules  
Therefore,  6.02  x  1023  molecules  x  10-­‐6  =  1  μmoles  

Avogadro  constant  6.02  x  1023    

One  gram  of  hydrogen  molecules  
12  grams  of  carbon  12  
X  grams  of  X  molecular  weigh  molecules  

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Absolute  amount   ConcentraMon  

moles                M    

Tris  molecular  weight  is  121.34  g  

⇒ 121.34  g  is  one  moles  
⇒ 121.34  g  (1moles)    in  1  L  is  1M  concentraMon  

Simple  calculaMon  method  

C1  x  V1=C2  x  V2  
ConcentraMon  (source)  X  Volume  (source)  =  ConcentraMon  (final)  X  Volume  (final)      

Make  10  mM  of    10  mL  Tris  soluMon      

Make  10  μM  of    10  mL  Tris  soluMon      

Storage  of  your  samples  

DNA  is  highly  stable    molecule,  but  avoid  followings  
 Highly  acidic  buffer  
 ContaminaMon  (by  other  samples,  bacterial  growth,  nucleases)  
 
RNA  is  highly  unstable  and  avoid  followings  
 UV,  or  highly  acidic  buffer  
 ContaminaMon  (by  other  samples,  bacterial  growth,  nucleases)  
 
***  RNase  is  highly  abundant  enzyme  in  everywhere  including  your  saliva  and  
fingerMps.    Most  of  all,  it  is  very  stable  enzymes  and  very  difficult  to  remove.    
Furthermore,  molecular  lab  frequently  use  Rnase  in  DNA  isolaMon.  

Enzymes  (proteins)  are  usually  in  30  to  50%  glycerol  stabilize  the  conformaMon  at  
-­‐20°C  without  freezing.    Once  you  dilute  it  in  a  regular  buffer,  avoid  to  freeze-­‐thaw.    Do  
not  vortex.  
 
Other  reagents  are  stored  according  to  the  manufacturer’s  instrucMon.    Very  oxen,  
you  may  make  aliquots  and  freeze  for  long  term  storage.  

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Working  with  nucleic  acid  

Images  from  Wikipedia  

Working  with  nucleic  acid  

The  way  to  indicate  the  direcMon  

of  DNA/RNA  is  always  5’  to  3’,  
which  is  also  the  direcMon  for  
polymerizaMon/translaMon.  

Images  from  Wikipedia  

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Chemistry  of  DNA  and  RNA  

Molecular  cloning  is  the  techniques  using  natural  

enzymes  for  modifying  the  DNA/RNA  for  your  study  

DNA  polymerase:    Taq  polymerase)  

DNA  ligase  
Topoisomerase  
RestricMon  enzymes:  EcoRI  (GAATTC)  
RNA  polymerase  
Images  from  Wikipedia  

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Molecular  cloning  is  the  techniques  using  natural  

enzymes  for  modifying  the  DNA/RNA  for  your  study  

hlp://cnx.org/contents/b3c1e1d2-­‐839c-­‐42b0-­‐a314-­‐e119a8aazdd@8.37:42  

Convenient  to  know:  IUPAC  notaMon  

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Those  nice  diagrams  showing  chemical  structures  of  nucleic  acids  

can  be  abstracted  to  a  simple  form,  in  parMcular  for  computer.  

ACTG  

Now,  lets  navigate  the  web  page  showing  standardized  ways  of  
expression  for  nucleic  acids  
 
Tribolium  castaneum  juvenile  hormone  acid  O-­‐methyltransferase  (Tcjhamt),  mRNA  
NM_001127311.1  
/translation="MNKASLYSKYSGLQKNDASFVIDNYLRLIKWKPNANILDIGSGD!
GNVIFELLLPKIPKHFAKFVGTDISEEMVLFAKNQCNDPKIDFLQMDISATIPPEFHE!
YFDHIFSFYCLHWVVEQRQAMKNIFDMLKPGGEMLLTFLASNPIYDIYERMAKSNKWG!
PYMNNLKKYISPYHHSEDPETELENLLKKEGFITHLCRVENRSYTFPSFSVLSKSVSA!
VNPFIKKLPENEIDTYIEDYLKEVRKLKTITIETCNNNDNEEKIHVPYKLFVTFASKP!
V"!
ORIGIN !
1 atgaacaaag cctcactgta ctcaaaatac agcggtttgc aaaaaaacga tgcgtctttt!
61 gtaatcgaca attacttgag actcatcaag tggaagccca acgcgaatat tttagacatc!
121 ggctcgggtg acggtaatgt aatattcgag cttttactcc cgaaaatccc caaacatttc!
181 gccaaattcg tcggaacgga catctccgaa gaaatggtcc tttttgcgaa aaatcagtgc!
241 aacgatccga aaatcgattt cctacaaatg gatatttcgg caacaattcc gcccgaattt!
301 cacgaatact tcgaccatat tttttcgttt tattgcttgc attgggtggt ggaacagagg!
361 caagccatga agaacatatt cgacatgctg aaaccggggg gcgaaatgct cctgactttc!
421 cttgccagca acccgattta tgacatttac gaacgaatgg ctaaatccaa caaatggggg!
481 ccatacatga acaatttaaa aaaatacatc tcgccctatc accattcgga ggatcctgaa!
541 accgagctgg agaatttgct gaaaaaggaa gggtttatta cgcatttgtg tcgagtggag!
601 aatcgctcgt acacttttcc cagtttttca gttttgtcaa aatcggtttc agcggtgaat!
661 ccgttcatta aaaaactgcc tgaaaacgaa attgacacct acattgagga ttacctcaaa!
721 gaggtcagga aactgaagac gatcacaatc gaaacgtgta acaataacga caatgaagag!
781 aaaatacacg tgccgtacaa actcttcgtt acatttgcct cgaaaccggt ctga!
//!

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What  do  we  do  today  

Groups  for  different  modules  

 Module  1:  Regular  PCR  
 Module  2:  Degenerate  PCR  
 
Opening  Christmas  presents  
 
Make  500mL  0.5x  TAE  buffer  
 
Make  primer  diluMons  for  100μL,  10μM  
 
Prepare  1%  agarose  gel  
 

Assignment  for  next  class  

Read  the  protocol  for  Qiagen  DNA  isolaMon  kit  

Pp.28-­‐30  are  the  protocol.  
 
Bring  your  samples,  
 Tribolium  freemany  for  module  1  
 Rhyzopertha  dominica  for  module  2  
 

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