Corum:DNA Hybridization

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Procedure)
m (Procedure)
Line 15: Line 15:
# Cool on bench 5 min.
# Cool on bench 5 min.
# Place weighted holder with sealed oligonucleotides into the beaker.
# Place weighted holder with sealed oligonucleotides into the beaker.
-
# Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into dsDNA linker at 1mM final concentration.
+
# Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into as single dsDNA linker with sticky ends at 1mM final concentration.
-
# Dilute the 1mM linker 1/10 (~10 μL 1mM linker into ~ 90 μL H<sub>2</sub>O, may need to be adjusted if some volume is lost and, for example only 9 μL 1mM linker is recovered). Mix and spin.
+
# Perform two 1/100 dilutions down to 100 nM (label all tubes clearly):
-
# Continue 1/10 dilutions down to 100 nM (label all tubes clearly):
+
#* 2 μL 1mM linker + 198 μL H<sub>2</sub>O = 200 μL 10μM linker
-
#* 20 μL 100μM linker + 180 μL H<sub>2</sub>O = 200 μL 10μM linker
+
#* 2 μL 10μM linker + 198 μL H<sub>2</sub>O = 200 μL 100nM linker
-
#* 20 μL 10μM linker + 180 μL H<sub>2</sub>O = 200 μL 1μM linker
+
# Store 1mM, 10μM, and 100nM stocks at -40 °C in latest "Primer" box. Keep an aliquot of the 100nM linker in your own box for use in constructions.
-
#* 20 μL 1μM linker + 180 μL H<sub>2</sub>O = 200 μL 100nM linker
+
-
# Store at -40 °C in latest "Primer" box.
+
==Notes==
==Notes==

Revision as of 19:22, 24 July 2012

SC 20:55, 19 June 2012 (EDT):

Contents

Overview

Hybridization of ssDNA oligonucleotides.

Materials

  • 5 μL 2mM sense oligonucleotide
  • 5 μL 2mM antisense oligonucleotide

Procedure

  1. Combine sense and antisense oligonucleotides. Vortex and spin.
  2. Seal tube airtight with paraffin and place in weighted holder.
  3. Boil 400 mL water in a 500 mL beaker.
  4. Cool on bench 5 min.
  5. Place weighted holder with sealed oligonucleotides into the beaker.
  6. Incubate on bench overnight or until water is about room temperature (~4 hr). At the end of this process, the complimentary ssDNA's are hybridized into as single dsDNA linker with sticky ends at 1mM final concentration.
  7. Perform two 1/100 dilutions down to 100 nM (label all tubes clearly):
    • 2 μL 1mM linker + 198 μL H2O = 200 μL 10μM linker
    • 2 μL 10μM linker + 198 μL H2O = 200 μL 100nM linker
  8. Store 1mM, 10μM, and 100nM stocks at -40 °C in latest "Primer" box. Keep an aliquot of the 100nM linker in your own box for use in constructions.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers, books, and websites

  1. New England Biolabs

Contact

or instead, discuss this protocol.

Personal tools