Daniel:Notebook/ComboLock/2016-8-5: Difference between revisions

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>Djacobse
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===Theory===
===Theory===


===Protocol===
C1 Amplicon: 5ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG3
 
C2 Amplicon: 5GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/3
 
LatchX1: 5CAAGATCATAAGAGTTCGTTCGTCCACCACACAGCGACGAGACCG3
 
Update: LatchX1 was a misorder. I need to use Latch X2 instead
 
LatchX2: 5CCCGATCACGCATCGCCGATACGGGCCATTTAGTGAACGCGACCGTCCACGG3
 
Reaction Trimer:
 
  LX2
C1|C2
                          3GGCACCTGCCAGCGCAAGTGATTTAC|CGGGCATAGCCGCTACGCACTAGCCC5   
ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/

Revision as of 18:03, 5 August 2016

Positive Control Amplicon Production

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In order to test the latch-padlock binding better, I am going to use a positive control oligonucleotide. The oligo is designed to contain only the parts of the C probes (and antibody oligos) that bind to the latch and padlock, as well as barcodes. The oligos will also have biotin, which means I'll have a good method for pulldown.

Oligonucleotide Design

First thing is to design the oligos. The following table indicates the created oligonucleotide sequences. The LatchX1, C1 amplicon and C2 amplicon were ordered from IDT. The reason for the additional latch is to remove the UMI on the normal latch sequences. Since the UMI is a degenerate sequence I can't design an oligo to bind to it. Therefore I replaced the UMI on the normal latch sequences with a second barcode for the LatchX1 probe. There are two amplicons because IDT doesn't allow you to attach a biotin to ultramers (>60 bp).

Name Sequence Reverse Complement Length (bp) Components
primer2 ACGGCGGACCTCGCACGG CCGTGCGAGGTCCGCCGT 18  
primer4 CCGTGGACGGTCGCGTTC GAACGCGACCGTCCACGG 18  
primer6 CGGCGATGCGTGATCGGG CCCGATCACGCATCGCCG 18  
primer12 CCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGG 18  
LatchX1 Barcode CATTTAGTATACGGGC GCCCGTATACTAAATG 16  
Latch 8 barcode TATTTGTA TACAAATA 8  
Latch 9 barcode GCGCCTAC GTAGGCGC 8  
Total Amplicon ACGGCGGACCTCGCACGGTATTT GTACCGTGGACGGTCGCGTTCGCCCGTATAC TAAATGCGGCGATGCGTGATCGGGGCG CCTACCCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGGGTAGG CGCCCCGATCACGCATCGCCGCATTTAGTAT ACGGGCGAACGCGACCGTCCACGGTAC AAATACCGTGCGAGGTCCGCCGT 104 Primer2-Latch8BC-Primer4-LatchX1BCRevComp-Primer6-Latch9BC-Primer12
C1 Amplicon ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG CATTTAGTGAACGCGACCGTCCACGGTACAAATACCGTGCGAGGTCCGCCGT 52  
C2 Amplicon GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGGGTAGGCGCCCCGATCACGCATCGCCGATACGGGC 52  
Latch X1 CAAGATCATAAGAGTTCGTTCGTCCACCACACAGCGACGAGACCG CGGTCTCGTCGCTGTGTGGTGGACGAACGAACTCTTATGATCTTG 45 primer6RC-bclp0001-bclp0002-primer4RC

Ligation Reaction

Since the amplicon is in pieces right now, I need to ligate the C1 and C2 amplicons together.

Theory

C1 Amplicon: 5ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG3

C2 Amplicon: 5GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/3

LatchX1: 5CAAGATCATAAGAGTTCGTTCGTCCACCACACAGCGACGAGACCG3

Update: LatchX1 was a misorder. I need to use Latch X2 instead

LatchX2: 5CCCGATCACGCATCGCCGATACGGGCCATTTAGTGAACGCGACCGTCCACGG3

Reaction Trimer:

 LX2
C1|C2
                         3GGCACCTGCCAGCGCAAGTGATTTAC|CGGGCATAGCCGCTACGCACTAGCCC5     
ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG|GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG/Biotin/