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Enzyme and Primer sequencing plates

Enzyme plates Probe/Primer plates PP-plate maps Notes  Comments

This protocol is for FRAGMENT library / M-P libraryM-P library / FRAGMENT library

/ M-P / FRAGMENT

35bpMate-Paired enzyme plates

1. check XStream program for 0.5ml Combitip:
   • setting: Dis
   • speed up: 1 (lowest)
   • speed down: 1 (lowest)
2. sign 5x 48-well plates for each machine: 2x "35A" and 3x "35C"2x "25A" and 3x "25C";
3. outline enzyme zones by fine color marker;
4. put all plates into big ice-box;
5. shortly vortex/spin-down and put in -20°C cold-box:
   1 run: 5 enzyme plates, 1 or 2 FlowCells

   1 FC 2 FC T4 DNA ligase 1.01ml 2.02ml Antarctic Phosphatase 264µl 528µl

   1 run: 10 enzyme plates, 1 or 2 FlowCells

   1 FC 2 FC T4 DNA ligase 1.42ml 2.84ml Antarctic Phosphatase 368µl 736µl

6. first dispense Antarctic Phosphatase (AP: 8µl), then T4 DNA ligase (L1: 10µl, L2: 20µl)
   
   
   #35A type (plates A and B for 35-bp run)
   

   ⁄ 1 2 3 4 5 6 7 8 A 1L1 1L2 2L1 2L2 1L1 1L2 2L1 2L2 B 3L1 3L2 4L1 4L2 3L1 3L2 4L1 4L2 C 5L1 5L2 6L1 6L2 5L1 5L2 6L1 6L2 D 7L1       7L1       E 1AP 2AP 3AP 4AP 1AP 2AP 3AP 4AP F 5AP 6AP     5AP 6AP       flowcell 1 flowcell 2

   
   
   
   #35C type (plates C, D and E for 35-bp run)
   

   ⁄ 1 2 3 4 5 6 7 8 A 1L1 1L2 2L1 2L2 1L1 1L2 2L1 2L2 B 3L1 3L2 4L1 4L2 3L1 3L2 4L1 4L2 C 5L1 5L2 6L1 6L2 5L1 5L2 6L1 6L2 D 7L1     BrL2 7L1     BrL2 E 1AP 2AP 3AP 4AP 1AP 2AP 3AP 4AP F 5AP 6AP   BrAP 5AP 6AP   BrAP   flowcell 1 flowcell 2

   #25A type (plates A and B for 25-bp run)
   

   ⁄ 1 2 3 4 5 6 7 8 A 1L1 1L2 2L1 2L2 1L1 1L2 2L1 2L2 B 3L1 3L2 4L1 4L2 3L1 3L2 4L1 4L2 C 5L1       5L1       D                 E 1AP 2AP 3AP 4AP 1AP 2AP 3AP 4AP F                   flowcell 1 flowcell 2

   
   
   
   #25C type (plates C, D and E for 25-bp run)
   

   ⁄ 1 2 3 4 5 6 7 8 A 1L1 1L2 2L1 2L2 1L1 1L2 2L1 2L2 B 3L1 3L2 4L1 4L2 3L1 3L2 4L1 4L2 C 5L1       5L1       D       BrL2       BrL2 E 1AP 2AP 3AP 4AP 1AP 2AP 3AP 4AP F       BrAP       BrAP   flowcell 1 flowcell 2

   
   * upper prefix is a ligation cycle



7. seal plate;
8. centrifuge at 4°C, 1000rpm, 10 sec;
9. keep at -20°C until use;





/ M-P / FRAGMENT

35bpMate-Paired Probe/Primer plates

10. check XStream program for 10ml Combitip:
    • setting: Dis
    • speed up: 5 (mid-range)
    • speed down: 3 (low-range)
11. (i) melt, (ii) vortex, (iii) spin-down and (iv) put in ice-box:

    1 FC 2 FC 5x T4 DNA ligase buffer 6.3ml 12.6ml 10x Phosphatase buffer 1.51ml 3.02ml mixA (Probe Mix A) 852µl 1.71ml mixB (Probe Mix B) 732µl 1.47ml FM2 (Focal Map P2 Label) 10µl 20µl P2B (P2 Block) 50.5µl 101µl 1A, 1B, 1C, 1D, 1E (Tag 1 Seq Primer A, B, C, D, E) 15µl each 30µl each B1C, B1D, B1E (Bridge Probe Tag 1 C, D, E) 48µl each 96µl each

    1 FC 2 FC 5x T4 DNA ligase buffer 8.9ml 17.8ml 10x Phosphatase buffer 2.1ml 4.2ml mixA (Probe Mix A) 1.25ml 2.5ml mixB (Probe Mix B) 1ml 2ml FM2 (Focal Map P2 Label) 20µl 40µl P1B (P1 Block) 50.5µl 101µl P2B (P2 Block) 50.5µl 101µl IA1 (IA Block 1) 50.5µl 101µl IA2 (IA Block 2) 50.5µl 101µl 1A, 1B, 1C, 1D, 1E (Tag 1 Seq Primer A, B, C, D, E) 15µl each 30µl each 2A1, 2B1, 2C1, 2D1, 2E1 (Tag 2 Seq Primer A1, B1, C1, D1, E1) 15µl each 30µl each 2A2, 2B2, 2C2, 2D2, 2E2 (Tag 2 Seq Primer A2, B2, C2, D2, E2) 15µl each 30µl each B1C, B1D, B1E (Bridge Probe Tag 1 C, D, E) 48µl each 96µl each B2C, B2D, B2E (Bridge Probe Tag 2 C, D, E) 48µl each 96µl each




12. prepare on NT:

    1 FC 2 FC H2O ~37ml ~75ml Sodium Acetate 1.4ml 2.8ml 20X SSPE 140µl 280µl

    1 FC 2 FC H2O ~53ml ~106ml Sodium Acetate ~2ml ~4ml 20X SSPE 280µl 560µl




13. prepare stock solutions (store them at 4°C)
    
    1 run: 5 plates (1FC) or 10 plates (2FC)1 run: 10 plates (1FC) or 20 plates (2FC)
    
    
    / M-P / FRAGMENT
    ◊   LP (Ligation Probe Mixture)

    per well 1 plate 10 plates x7.2 wells x71 wells H2O 306µl 2.203ml 3x 734µl 21.73ml 5x T4 DNA ligase buffer 90µl 648µl 6.39ml Sodium Acetate 20µl 144µl 1.42ml mixA, 1mM 24µl 173µl 1.7ml Total: 440µl 3.17ml 31.24ml

    per well 1 plate 10 plates x5.2 wells x51 wells H2O 306µl 1.591ml 2x 780µl 15.606ml 5x T4 DNA ligase buffer 90µl 468µl 4.59ml Sodium Acetate 20µl 104µl 1.02ml mixA, 1mM 24µl 125µl 1.224ml Total: 440µl 2.288ml 22.44ml

    
    
    
    / M-P / FRAGMENT
    ◊   P3 (Probe Mix B Ligation Mixture)

    per well 1 plate 10 plates x6.2 wells x61 wells H2O 296µl 1.835ml 18.06ml 5x T4 DNA ligase buffer 90µl 558µl 5.49ml Sodium Acetate 20µl 124µl 1.22ml mixB, 1mM 24µl 149µl 1.46ml Total: 430µl 2.67ml 26.23ml

    per well 1 plate 10 plates x4.2 wells x41 wells H2O 296µl 1.243ml 12.136ml 5x T4 DNA ligase buffer 90µl 378µl 3.69ml Sodium Acetate 20µl 84µl 0.82ml mixB, 1mM 24µl 101µl 0.984ml Total: 430µl 1.806ml 17.63ml

    
    
    
    / M-P / FRAGMENT
    ◊   PB (Phosphatase Buffer)

    per well plates A & B plates C, D & E all 5 plates x6.2 wells x7.2 wells x33.5 wells H2O 397µl 2.461ml 2.858ml 13.3ml 10x Phosphatase buffer 45µl 279µl 324µl 1.508ml Total: 442µl 2.74ml 3.18ml 14.81ml

    per well plates A & B plates C, D & E all 5 plates x4.2 wells x5.2 wells x23.5 wells H2O 397µl 1.667ml 2.064ml 9.33ml 10x Phosphatase buffer 45µl 189µl 234µl 1.058ml Total: 442µl 1.856ml 2.298ml 10.387ml

    
    
    ◊   PS (Pre-scan Reagent Mixture)

    per well 2x FC 4x FC x2.02 wells x4.05 wells H2O 417µl 842µl 1.689ml 20X SSPE Buffer 23µl 46.5µl 93µl FM2, 200µM 10µl 20µl 40.5µl Total: 450µl 909µl 1.82ml

    
    
    
    / M-P / FRAGMENT
    ◊   P-XF-X & R-X (Priming Oligo Mixtures)
    for two FC's coefficient is: 2.02x; for four: 4.05x
    FRAGMENT library
    number of FC's:

    Component #P-A #P-B #P-C #P-D #P-E #A-E H2O 20X SSPE Buffer P2 block Tag 1 Seq Primer 1A:  1B:  1C:  1D:  1E:    Total:

    
    Mate-paired library Tag1 (Forward)
    number of FC's:

    Component #F-A #F-B #F-C #F-D #F-E #A-E H2O 20X SSPE Buffer IA block 1 IA block 2 P2 block Tag 2 Seq Primer 1A:  1B:  1C:  1D:  1E:    Total:

    
    
    Mate-paired library Tag2 (Rewerse)
    number of FC's:

    Component #R-A #R-B #R-C #R-D #R-E #A-E H2O 20X SSPE Buffer P1 block P2 block Tag 1 Seq Primer 2A1:  2A2:  2B1:  2B2:  2C1:  2C2:  2D1:  2D2:  2E1:  2E2:    Total:

    
    
    
    / M-P / FRAGMENT
    ◊   B1-C, B1-D & B1-EBX-C, BX-D & BX-E (Bridge Probes)
    for two FC's coefficient is: 2.02x; for four: 4.05x
    
    number of FC's:

    Component #B1-C #B1-D #B1-E #C-E H2O 5x T4 DNA ligase buffer Sodium Acetate Solution Bridge Probe Tag 1 B1C:  B1D:  B1E:    Total:

    Mate-paired library Tag1 (F3)
    number of FC's:

    Component #B1-C #B1-D #B1-E #C-E H2O 5x T4 DNA ligase buffer Sodium Acetate Solution Bridge Probe Tag 1 B1C: B1D: B1E:   Total:

    Mate-paired library Tag2 (R3)
    number of FC's:

    Component #B2-C #B2-D #B2-E #C-E H2O 5x T4 DNA ligase buffer Sodium Acetate Solution Bridge Probe Tag 2 B2C:    B2E:    Total:




/ M-P / FRAGMENT
14. put reagent into appropriate plates using XStream with 10ml Combitip:

    Component A B C D E LP 7x 440µl P3 6x 430µl PB 6x 440µl 7x 440µl PS 1x 450µl ----- #PO-A, -B, -C, -D, -E 1x 450µl #BP-C, -D, -E ----- 1x 430µl

    Component Forward run Reverse run 1A 1B 1C 1D 1E 2A 2B 2C 2D 2E LP 5x 440µl 5x 440µl P3 4x 430µl 4x 430µl PB 4x 440µl 5x 440µl 4x 440µl 5x 440µl PS 1x 450µl ----- 1x 450µl ----- #F-A, -B, -C, -D, -E 1x 450µl ----- #R-A, -B, -C, -D, -E ----- 1x 450µl #B1-C, -D, -E ----- 1x 430µl ----- #B2-C, -D, -E ----- ----- 1x 430µl




15. seal plates;
16. if it will be used within 4 days, keep plate at 4°C, otherwise, snap freeze in liquid nitrogen and keep at -20°C;




/ M-P / FRAGMENT

PP-plate maps

F35, A(1)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                       6PB D                       5PB E                       4PB F                       3PB G 1P3 2P3 3P3 4P3 5P3 6P3           2PB \ H 1LP 2LP 3LP 4LP 5LP 6LP 7LP     0PS 0P-A 1PB



F35, B(2)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                       6PB D                       5PB E                       4PB F                       3PB G 1P3 2P3 3P3 4P3 5P3 6P3           2PB \ H 1LP 2LP 3LP 4LP 5LP 6LP 7LP       0P-B 1PB



F35, C(3)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                       6PB D                       5PB E                       4PB F                       3PB G 1P3 2P3 3P3 4P3 5P3 6P3     0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP 6LP 7LP   0B1-C   0P-C 1PB



F35, D(4)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                       6PB D                       5PB E                       4PB F                       3PB G 1P3 2P3 3P3 4P3 5P3 6P3     0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP 6LP 7LP   0B1-D   0P-D 1PB



F35, E(5)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                       6PB D                       5PB E                       4PB F                       3PB G 1P3 2P3 3P3 4P3 5P3 6P3     0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP 6LP 7LP   0B1-E   0P-E 1PB

Forward run

Forward, A(1)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3               2PB \ H 1LP 2LP 3LP 4LP 5LP         0PS 0F-A 1PB



Forward, B(2)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3               2PB \ H 1LP 2LP 3LP 4LP 5LP           0F-B 1PB



Forward, C(3)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B1-C   0F-C 1PB



Forward, D(4)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B1-D   0F-D 1PB



Forward, E(5)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B1-E   0F-E 1PB

Reverse run

Reverse, A(1)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3               2PB \ H 1LP 2LP 3LP 4LP 5LP         0PS 0R-A 1PB



Reverse, B(2)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3               2PB \ H 1LP 2LP 3LP 4LP 5LP           0R-B 1PB



Reverse, C(3)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B2-C   0R-C 1PB



Reverse, D(4)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B2-D   0R-D 1PB



Reverse, E(5)

1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                       4PB F                       3PB G 1P3 2P3 3P3 4P3         0PB     2PB \ H 1LP 2LP 3LP 4LP 5LP       0B2-E   0R-E 1PB






Notes




Second-generation sequencing
URL: http://seq.zbio.net
e-mail: soldatov@molgen.mpg.de
visits:
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