Paul.xu at 19:09, 4 February 2018

2018-02-04T19:09:41Z

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== Oligo Primer Preparation ==

'''100 uM:'''

for example:

Resuspend the 42.6nmol of primer in 426 ul EB or H2O=100 uM

ug= (umole*molecular weight)/1000

for example

42.6 nmole, 325.7ug, add 426 ul H2O to make 100 uM

1 ul 100 uM stock-->325.7ug/426ul=0.76ug/ul

10 ul of 10 uM stock-->0.76 ug/ul

== Phosphorylation With T4 PNK ==

1X T4 Polynucleotide Kinase Reaction Buffer: 70 mM Tris-HCl 10 mM MgCl2 5 mM dithiothreitol pH 7.6 @ 25°C

{|
|-
| T4 PNK
| 1 µl (10 units)
|-
| 10X T4 PNK Buffer
| 5 µl
|-
| 10 mM ATP
| 5 µl (1 mM final conc.)
|-
| DNA (e.g., 25 mer primers)
| 1-2 µg, 20 ul of 10 uM stock
|-
| Nuclease-free Water
| Add 19 ul H2O to 50 µl final vol
|-
| Incubation
| 37°C for 30 minutes
|}

Final oligo conc is 4 uM

For PCR: 2.5 ul each of phosphorylated F and R.

=== Phosphorylation using T4 DNA Ligase ===

'''Use 100 uM stock'''

100 µM oligo stock 2 µL 

10X T4 DNA ligase buffer 2 µL 

Sterile water 15 µL 

T4 Polynucleotide Kinase 1 µL 

Total 20 µL 

'''Use 10 uM stock'''

10 µM oligo stock 17 µL 

10X T4 DNA ligase buffer 2 µL 

T4 Polynucleotide Kinase 1 µL 

Total 20 µL 

Incubate the reaction mixture at 37 oC for 1 hour and heat inactivate the T4 PNK at 65 oC for 20 minutes. Store the phosphorylated oligos at -20 oC till further use.

== Oligonucleotide phosphorylation and annealing ==

Oligonucleotides (oligos) come in tubes (dehydrated) or as liquid stocks (may only be available in 96-well plate format).

De-hydrated oligos will have a specified quantity (usually in nanomoles) on the tube.

Reconstitute the dehydrated oligos with sterile (or RNAase, DNAase free) water to a 100 µM stock. As a rule of thumb, add (ten times the number of nanomoles of DNA in the tube) µL water to make a 100 µM stock.

'''Oligo phosphorylation:'''

Set up separate phosphorylation reactions for each forward (FW) and reverse (RV) oligos

100 µM oligo stock 2 µL

10X T4 DNA ligase buffer 2 µL

Sterile water 15 µL

T4 Polynucleotide Kinase 1 µL

Total 20 µL

Incubate the reaction mixture at 37 0 C for 1 hour and heat inactivate the T4 PNK at 65 0 C for 20 minutes. Store the phosphorylated oligos at -20 0 C till further use. •

NEB PNK enzyme is supplied with its PNK buffer and it does not contain the ATP required for the phosphorylation reaction. To simplify the reaction step, use T4 ligase buffer (instead of PNK buffer). T4 ligase buffer provides the appropriate amount (1mM ATP) of phosphate for the phosphorylation reaction. • Use fresh T4 ligase buffer (<1 year old) and avoid repeated freeze/thaw cycles of the buffer. The DTT present in T4 ligase buffer oxides naturally and its oxidation is accelerated by repeated freeze/thaw cycles or excessive heating. Annealing the phosphorylated FW and RV oligos: FW oligo 5 µL RV oligo 5 µL Sterile water 90 µL Total 100 µL

Annealing the phosphorylated FW and RV oligos: FW oligo 5 µL RV oligo 5 µL Sterile water 90 µL Total 100 µL Incubate the phosphorylated oligos at 95 oC for 3 minutes. If handling >10 annealing reactions, using a thermocycler is convenient. In that case, choose a program step without a heated lid (to ease removal). Cool the reaction slowly at room temperature for ~ 30 min to 1 hr, check the concentration with a nanodrop and store the annealed products at -20 oC. 
[[Category:Protocols]]

T4 PNK - Revision history

Paul.xu at 19:09, 4 February 2018