Annealing primers

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 1: Line 1:
-
A simple and cheap way to make a short (<100bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. 
+
=Annealing complementary primers=
-
*When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/<math>\mu</math>l.
+
A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. 
 +
 
 +
*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/&mu;l.
*For the annealing mix one recipe that works is as follows -  
*For the annealing mix one recipe that works is as follows -  
-
**4<math>\mu</math>l of each of the concentrated primers.
+
**4 &mu;L of each of the concentrated primers.
-
**4<math>\mu</math>l of salt solution (10mM NaCl)
+
**4 &mu;L of salt solution (10 mM NaCl)
-
**28<math>\mu</math>l of water
+
**28 &mu;L of water
*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
-
*Anneal the primers by heating them at least 5<sup>o</sup>C above their melting point and cooling them down slowly in stages using a [[Thermocycler]].  Melting Temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.
+
*Anneal the primers by heating them at least 5&deg;C above their melting point and cooling them down slowly in stages using a [[Thermocycler]].  Melting temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.
*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.  Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.
*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.  Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.
Line 16: Line 18:
*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
-
*If you are interested in cloning piece of DNA and include restriction enzyme recognition sites near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA. So include extra bases to increase the efficiency of cutting.  Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully).  Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here]. Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
+
=Annealing and primer extension=
 +
 
 +
This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp).  The DNA fragment is prepared for cloning by restriction digest.
 +
 
 +
==Materials==
 +
 
 +
*Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below.  See [[Restriction Digest]] for notes on cutting near the ends of linear DNA fragments.
 +
 
 +
Oligo 1: &nbsp;&nbsp;&nbsp;5' ---RE site-------------------------------- 3'<br>
 +
Oligo 2:    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3' --------------------------------RE site--- 5'
 +
 
 +
*[http://www.neb.com/nebecomm/products/productM0212.asp Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase]
 +
*dNTPs (25 mM each dNTP in stock)
 +
*Restriction enzyme(s)
 +
*Restriction enzyme buffer
 +
*BSA
 +
 
 +
==Calculating amount of oligo for reaction==
 +
 
 +
''This should be checked for errors'' -[[User:Rshetty|Reshma]] 19:03, 12 May 2005 (EDT)
 +
 
 +
<math> \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}</math>
 +
 
 +
==Procedure==
 +
 
 +
#Dilute the two oligos to a concentration of 10 or 25 &mu;M using H<sub>2</sub>O
 +
#Mix the following in a 0.6 mL sterile tube
 +
#*10 &mu;L 10X restriction enzyme buffer
 +
#*1 &mu;L 100X BSA
 +
#*X &mu;L oligo 1 (typically 1 &mu;g or more)
 +
#*Y &mu;L oligo 1 (typically 1 &mu;g or more)
 +
#*(87 - 2X) &mu;L deionized sterile H<sub>2</sub>O
 +
#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94&deg;C for 5 mins followed by a cool down for 0.1&deg;C/sec to 37&deg;C or by placing the tube in a beaker of boiling water and letting it cool to room temperature.
 +
#Add 1 &mu;L Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.
 +
#Add 1 &mu;L dNTPS (equal to 0.25 mM final concentration of each dNTP) <br> ''Recommend using a thermal cycler for the following incubation steps.''
 +
#Incubate 1 hr at 37&deg;C.
 +
#Heat inactivate polymerase by incubating at 75&deg;C for 20 minutes. <br> ''See [[Restriction Digest]] for more information on the following steps.''
 +
#Add 1 &mu;L restriction enzyme(s) to mixture.  
 +
#Incubate for a minimum of 2 hrs.
 +
#Heat inactivate restriction enzyme by incubating at 80&deg;C for 20 mins.
 +
#[[Purification of DNA | Purify DNA]] as necessary

Revision as of 19:03, 12 May 2005

Contents

Annealing complementary primers

A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as Invitrogen.

  • When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl.
  • For the annealing mix one recipe that works is as follows -
    • 4 μL of each of the concentrated primers.
    • 4 μL of salt solution (10 mM NaCl)
    • 28 μL of water
  • The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
  • Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a Thermocycler. Melting temperature calculations can best be done using software such as VectorNTI or data may come with the primers themselves.
  • A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100oC and certainly any non-specific binding should be melted at that temperature.
  • Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as PNK Treatment of DNA Ends

Annealing and primer extension

This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.

Materials

  • Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below. See Restriction Digest for notes on cutting near the ends of linear DNA fragments.

Oligo 1:    5' ---RE site-------------------------------- 3'
Oligo 2:                                        3' --------------------------------RE site--- 5'

Calculating amount of oligo for reaction

This should be checked for errors -Reshma 19:03, 12 May 2005 (EDT)

 \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}

Procedure

  1. Dilute the two oligos to a concentration of 10 or 25 μM using H2O
  2. Mix the following in a 0.6 mL sterile tube
    • 10 μL 10X restriction enzyme buffer
    • 1 μL 100X BSA
    • X μL oligo 1 (typically 1 μg or more)
    • Y μL oligo 1 (typically 1 μg or more)
    • (87 - 2X) μL deionized sterile H2O
  3. Anneal the two oligos together by either placing the mixture in a thermal cycler (MJ Research, PTC-200) at 94°C for 5 mins followed by a cool down for 0.1°C/sec to 37°C or by placing the tube in a beaker of boiling water and letting it cool to room temperature.
  4. Add 1 μL Klenow 3'\rightarrow5' exo- polymerase to mixture.
    Vortex polymerase before pipetting to ensure it is well-mixed.
  5. Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP)
    Recommend using a thermal cycler for the following incubation steps.
  6. Incubate 1 hr at 37°C.
  7. Heat inactivate polymerase by incubating at 75°C for 20 minutes.
    See Restriction Digest for more information on the following steps.
  8. Add 1 μL restriction enzyme(s) to mixture.
  9. Incubate for a minimum of 2 hrs.
  10. Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
  11. Purify DNA as necessary
Personal tools