From: CAMPBELL@EARN.FRCICT81
Newsgroups: bionet.molbio.methds-reagnts
Subject: library screening
Date: 28 Jul 92 10:34:39 GMT
Distribution: bionet
P1-Message-Id: FR*ATLAS*CICT; 920728104127796-CICT-TLSE
Original-To: methods@UK.AC.DARESBURY
 FR*ATLAS*CICT; arrival Tue, 28 Jul 92 11: 34:39 +0100 action Relayed
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Fellow netters:
                                                                                                                                                                                                                                                          
A couple of fundamental questions regarding cDNA library screening...                                                                                                                                                                                     
                                                                                                                                                                                                                                                          
First some background:                                                                                                                                                                                                                                    
I have in hand four 'pools' of degenerate oligos derived from amino acid                                                                                                                                                                                  
sequence from a protein that I purified.  Each pool is derived from a                                                                                                                                                                                     
different peptide sequence and the degeneracy of each pool ranges from                                                                                                                                                                                    
64 to 1024 potential sequences.  Pool one is derived from a sequence of                                                                                                                                                                                   
32 nt (10 degenerate positions), pool two is 29 nt long (9 degenerate                                                                                                                                                                                     
positions), pool 3 is 25 nt long (8 degenerate positions), and pool 4 is                                                                                                                                                                                  
30 nt long (6 degenerate positions).  The high level of degeneracy                                                                                                                                                                                        
arises from the fact that 1) my peptides contained a lot of amino acids                                                                                                                                                                                   
which are encoded by two or more codons and 2) I have very limited codon                                                                                                                                                                                  
usage data with which to design oligos from aa sequence data in my species                                                                                                                                                                                
(Eucalyptus).  Nonetheless, I feel that I have designed the best oligos                                                                                                                                                                                   
possible and would like to begin screening our cDNA library for a clone.                                                                                                                                                                                  
                                                                                                                                                                                                                                                          
I intend to use 3' oligo tailing with Digoxigenin-dUTP to label my oligos                                                                                                                                                                                 
and, due to the degenrate nature of my oligos, use base composition-                                                                                                                                                                                      
independent hybridization with tetramethylammonium chloride (Wood et al                                                                                                                                                                                   
(1985) PNAS 82: 1585-1588) to screen the library and finally chemiluminescent                                                                                                                                                                             
detection.  My problem lies in the fact that I have no experience with                                                                                                                                                                                    
3' tailing and tetramethylammonium chloride hybridization.                                                                                                                                                                                                
                                                                                                                                                                                                                                                          
I am planning to run a pilot _Northern_ blot with the RNA that was used to                                                                                                                                                                                
generate the cDNA library in order to determine: 1) the best conditions                                                                                                                                                                                   
for hybridization; 2) which pool gives me best (strongest?) hybridization                                                                                                                                                                                 
signal; 3) if all pools light up the same molecular weight band (I sure hope                                                                                                                                                                              
so).                                                                                                                                                                                                                                                      
                                                                                                                                                                                                                                                          
Now my questions:                                                                                                                                                                                                                                         
                                                                                                                                                                                                                                                          
1) Are the results that one obtains with Northern blots (in terms of best                                                                                                                                                                                 
   hybridization conditions) transferable to plaque lifts when one is using                                                                                                                                                                               
   tetramethylammonium chloride? (I would assume so given that the hyb is                                                                                                                                                                                 
   base-independent)                                                                                                                                                                                                                                      
                                                                                                                                                                                                                                                          
2) Is there a way of determining which pool of oligos to use in library                                                                                                                                                                                   
   screening? (I was going to use my least degenerate pool first and then                                                                                                                                                                                 
   use sequentially more degenerate pools in the subsequent rounds  of                                                                                                                                                                                    
   plaque selection; however, I thought that the pilot experiment with                                                                                                                                                                                    
   the Northern blot might suggest a different strategy).                                                                                                                                                                                                 
                                                                                                                                                                                                                                                          
3)  What is the best tail length for 3' tailed oligos when screening                                                                                                                                                                                      
    cDNA libraries?  How badly can the tail length skew hybridization                                                                                                                                                                                     
    conditions such that one ends up selecting false positives? (I'm                                                                                                                                                                                      
    including poly A and poly dA in the hyb buffer to try to circumvent                                                                                                                                                                                   
    such problems  but perhaps a more judicious choice of tail length                                                                                                                                                                                     
    would be better, oui?)                                                                                                                                                                                                                                
                                                                                                                                                                                                                                                          
4)  Does anyone have experience with both 3' tailing and tetramethylammonium                                                                                                                                                                              
    chloride in cDNA screening?  Any trouble shooting advice?                                                                                                                                                                                             
                                                                                                                                                                                                                                                          
All criticisms, comments and advice warmly received (of course, comments and                                                                                                                                                                              
advice received more warmly than criticisms ;-)).                                                                                                                                                                                                         
                                                                                                                                                                                                                                                          
Thanks in advance.                                                                                                                                                                                                                                        
                                                                                                                                                                                                                                                          
Malcolm                                                                                                                                                                                                                                                   
                                                                                                                                                                                                                                                          
Malcolm M. Campbell, Ph.D.                                                                                                                                                                                                                                
Equipe lignines                                                                                                                                                                                                                                           
Centre de Physiologie Vegetale                                                                                                                                                                                                                            
Universite Paul Sabatier                                                                                                                                                                                                                                  
Toulouse, FRANCE                                                                                                                                                                                                                                          
                                                                                                                                                                                                                                                          
CAMPBELL@FRCICT81.BITNET                                                                                                                                                                                                                                  
                                                                                                                                                                                                                                                          
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US Vice President Dan Quayle explaining to a grade school spelling bee                                                                                                                                                                                    
participant how to spell tomato (or perhaps it was potato)                                                                                                                                                                                                
                                                                                                                                                                                                                                                          
Tomatoe, tomatto                                                                                                                                                                                                                                          
Potatoe, pottato                                                                                                                                                                                                                                          
Let's Quayle the whole thing off                                                                                                                                                                                                                          
                                                                                                                                                                                                                                                          
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