Endy:Double stranding oligo libraries

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m (Separate on a gel and do a second PCR cleanup)
Current revision (15:39, 29 August 2006) (view source)
(Perform PCR cleanup on the double-stranded library)
 
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==Order oligos and double-stranding [[Designing_primers|primers]]==
==Order oligos and double-stranding [[Designing_primers|primers]]==
* Dilute stocks to 100uM
* Dilute stocks to 100uM
-
* Dilute sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)
+
* Dilute working stocks of libraries and double-stranding primers to 10uM
-
* Dilute double-stranding primers to 10uM
+
* Dilute working stocks of sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)
* Some considerations:
* Some considerations:
** Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency
** Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency
-
** Make sure you have some spacer sequence around the restriction site.  [http://www.neb.com/ NEB] has a list of the length of the spacer sequence required for each restriction enzyme.
+
** Make sure you have some spacer sequence around the restriction site.  [http://www.neb.com/ NEB] has a list of the length of the spacer sequence required for each restriction enzyme. ([[Restriction digest#Notes|8bp is usually a safe bet]])
** Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo.  This will already be more than you’ll need.
** Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo.  This will already be more than you’ll need.
** If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc.  This allows for you to generate a library which is much more likely to produce productive clones.
** If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc.  This allows for you to generate a library which is much more likely to produce productive clones.
==Double strand the library with modified PCR==
==Double strand the library with modified PCR==
-
* Total library DNA should be <25pmol per 100uL reaction
+
*Expected max library size is 10<sup>8</sup> molecules (limit set by transformation efficiency.)  You want to load 10X the expected library size for a single library construction.  Therefore, you would like to have 10<sup>9</sup> molecules for a single transformation.
-
* You want to start with 10X the final desired amount of library for PCR
+
**1pmol corresponds to ~10<sup>11</sup> molecules
-
** Split into separate 100uL reactions if necessary
+
**Use 25pmol of library to make enough for 2500 transformations
 +
*Total library DNA should be less than ~25pmol per 100uL reaction
-
===Reaction Mix (100uL)===
+
===Reaction Mix (100uL, 25pmol library)===
Use the following reaction mix for each PCR reaction:
Use the following reaction mix for each PCR reaction:
*10 &mu;l 10x Thermo polymerase buffer
*10 &mu;l 10x Thermo polymerase buffer
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*2.5 &mu;l 10&mu;M library stock
*2.5 &mu;l 10&mu;M library stock
-
===PCR protocol=== ???
+
===PCR protocol===
-
*95 C for 2.5 minutes
+
*95<sup>o</sup>C for 2.5 minutes
*Cycle 5 times:
*Cycle 5 times:
-
**53 C (or whatever temperature is appropriate) for 30 s (annealing)  
+
**55<sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)  
-
**72 C for 30 s (elongation)
+
**72<sup>o</sup>C for 1.5 minutes (elongation)
-
*72 C for 10 minutes (final elongation)
+
*72<sup>o</sup>C for 10 minutes (final elongation)
-
*4 C forever
+
*4<sup>o</sup>C forever
-
==PCR cleanup on the double-stranded libraries==
+
==Perform PCR cleanup on the double-stranded library==
* This concentrates the samples and allows for the buffer to be switched to something more appropriate.
* This concentrates the samples and allows for the buffer to be switched to something more appropriate.
-
* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug)
+
* PCR purification columns can handle up to 10ug of DNA
 +
** 100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column
* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
 +
* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded) [[Image:Double-stranded_oligo_libraries.jpg|thumb|none|300px|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]
-
==[[Restriction digest]] the libraries==
+
==[[Restriction digest]] the library==
-
==Separate on a [[Agarose_gel_electrophoresis|gel]] and do a second PCR cleanup==
+
==Perform PCR cleanup on the digest==
-
* Alternatively, you can run a sample of the first PCR reaction out on a gel for analysis against a sample of the original library (double stranded should run slightly faster than single stranded), then perform the digest.  Doing a PCR cleanup on the digest will remove the cut ends, since they are small.
+
* This will remove the cut ends, since they are small.
==[[DNA_Ligation|Ligate]] the sample from the PCR cleanup with a vector==   
==[[DNA_Ligation|Ligate]] the sample from the PCR cleanup with a vector==   
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* This will either be done via [[Electroporation|electroporation]] or [[Transforming_chemically_competent_cells|chemically compotent cells]], we’re experimenting now to see which one is more efficient.
* This will either be done via [[Electroporation|electroporation]] or [[Transforming_chemically_competent_cells|chemically compotent cells]], we’re experimenting now to see which one is more efficient.
-
==Notes==
+
[[Category:Protocol]]
-
???
+
[[Category:DNA]]
-
Expected max library concentration is 10^8 molecules (this is a limit set by the transformation efficiency.)  So for step 2, you would like to have 10^9 molecules for a single library transformation (more can be used so a stock can be kept.)
+
[[Category:In vitro]]
-
We will typically want a library to have approximately 10^10 molecules (~0.1pmol)
+

Current revision

Contents

Order oligos and double-stranding primers

  • Dilute stocks to 100uM
  • Dilute working stocks of libraries and double-stranding primers to 10uM
  • Dilute working stocks of sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)
  • Some considerations:
    • Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency
    • Make sure you have some spacer sequence around the restriction site. NEB has a list of the length of the spacer sequence required for each restriction enzyme. (8bp is usually a safe bet)
    • Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo. This will already be more than you’ll need.
    • If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc. This allows for you to generate a library which is much more likely to produce productive clones.

Double strand the library with modified PCR

  • Expected max library size is 108 molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 109 molecules for a single transformation.
    • 1pmol corresponds to ~1011 molecules
    • Use 25pmol of library to make enough for 2500 transformations
  • Total library DNA should be less than ~25pmol per 100uL reaction

Reaction Mix (100uL, 25pmol library)

Use the following reaction mix for each PCR reaction:

  • 10 μl 10x Thermo polymerase buffer
  • 10 μl 10x dNTPs (10x = 2.5 mM each dNTP)
  • 5 μl 10 μM FWD primer
  • 5 μl 10 μM REV primer
  • 1 μl Polymerase (taq or vent)
  • 66.5 μl H2O
  • 2.5 μl 10μM library stock

PCR protocol

  • 95oC for 2.5 minutes
  • Cycle 5 times:
    • 55oC (or whatever temperature is appropriate) for 30 seconds (annealing)
    • 72oC for 1.5 minutes (elongation)
  • 72oC for 10 minutes (final elongation)
  • 4oC forever

Perform PCR cleanup on the double-stranded library

  • This concentrates the samples and allows for the buffer to be switched to something more appropriate.
  • PCR purification columns can handle up to 10ug of DNA
    • 100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column
  • Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
  • You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)
    Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)
    Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)

Restriction digest the library

Perform PCR cleanup on the digest

  • This will remove the cut ends, since they are small.

Ligate the sample from the PCR cleanup with a vector

Transform into compotent cells

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