5´ DNA Adenylation Kit

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
E2610S     -20       Mth RNA Ligase M2611AVIAL -20 1 x 0.02 ml 50 µM   5' DNA Adenylation Reaction Buffer B2610SVIAL -20 1 x 1 ml 10 X   Adenosine 5' Triphosphate N0757AVIAL -20 1 x 0.1 ml 1 mM
E2610L     -20       Mth RNA Ligase M2611AAVIAL -20 1 x 0.1 ml 50 µM   5' DNA Adenylation Reaction Buffer B2610SVIAL -20 1 x 1 ml 10 X   Adenosine 5' Triphosphate N0757AVIAL -20 1 x 0.1 ml 1 mM

Features

• One step reaction gives quantitative adenylation
• Simpler than existing chemical and enzymatic methods
• Reduces need for extensive purification of reaction product
• 65°C reaction temperature reduces secondary structural concerns
• Easily scalable from pmol to μmol range

Application Features

Enzymatic 5´-adenylation of single-stranded DNA linkers for next generation sequencing.

1. For substrates with unprotected 3´ termini increase concentration of ATP to 0.5 mM to prevent circularization and concatemerization.
2. The low turnover of the enzyme requires an approximately equimolar concentration of the enzyme and the oligonucleotide substrate.
3. Adenylated DNA linkers can be used for 3´ end ligation of RNA in cDNA library preparation for Next Generation sequencing protocols [3,4].

1. Zhelkovsky, A.M. and McReynolds, L.A. (2011). Nucl. Acids Res. 39(17): e117.
2. Torchia, C., Takagi, Y. and Ho, C.K. (2008). Nucleic Acids Res. 36, 6218-6227.
3. Hafner, M. et al. (2008). Methods. 44, 3-12.
4. Vigneault, F., Sismour, A.M. and Church, G.M. (2008). Nature Methods. 5, 777-779.

1. Protocol for Oligonucleotide Adenylation (NEB #E2610)
2. Protocol for one-step adenylation and ligation of 5´p DNA and 3´OH RNA using 5´ DNA Adenylation Kit (NEB #E2610) and Thermostable 5´ App DNA/RNA Ligase (NEB #M0319)

• Properties of DNA and RNA Ligases
• RNA Ligase Selection Chart

1. Can this 5' DNA adenylation kit be used for adenylating ssRNA oligos?
2. What is the molecular weight of Mth RNA Ligase? Is it a fusion protein?
3. What ratio of enzyme to oligonucleotide do you recommend
4. Can Mth RNA Ligase be used in other NEBuffers?
5. Can Mth RNA Ligase be used for ligation?
6. Does PEG stimulate adenylation?
7. What concentration of ATP should be used in the adenylation reaction?
8. Will the enzyme work at low temperature (e.g., 37°C)?
9. How do I determine the extent of adenylation?
10. Do I need to clean up the adenylation reaction before I use the oligo for ligation?
11. What is the composition of the 5´ DNA Adenylation Reaction Buffer at 1X concentration?
12. What is the salt tolerance of this RNA ligase?
13. How can I scale up my adenylation reaction?

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

• E2610S_L_v1

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

• E2610S_L_v1_0031708
• E2610S_v1_10009214
• E2610L_v1_10009213
• E2610S_v1_10028389
• E2610L_v1_10028388
• E2610L_v1_10040004
• E2610L_v1_10043152
• E2610L_v1_10049235
• E2610S_v1_10049236
• E2610S_v1_10053874
• E2610L_v1_10053873
• E2610S_v1_10060670
• E2610L_v1_10062831
• E2610L_v1_10068001
• E2610S_v1_10068000
• E2610L_v1_10081110
• E2610L_v1_10091344
• E2610S_v1_10091345
• E2610L_v1_10102426
• E2610S_v1_10102427
• E2610L_v1_10118370
• E2610L_v1_10124259
• E2610S_v1_10136950
• E2610S_v1_10144335
• E2610L_v1_10144336
• E2610S_v1_10148951
• E2610S_v1_10172043
• E2610S_v1_10176041
• E2610L_v1_10179953
• E2610S_v1_10180291
• E2610S_v1_10195957
• E2610S_v1_10204825
• E2610L_v1_10220918
• E2610S_v1_10226296
• E2610L_v1_10243731
• E2610S_v1_10266645
• E2610L_v1_10270615
• E2610S_v1_10293093
• E2610L_v1_10308193
• E2610S_v1_10308824
• E2610L_v1_10313589
• E2610L_v1_10324759
• E2610S_v1_10338782

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• Mth RNA Ligase
• 5' DNA Adenylation Reaction Buffer
• Adenosine 5' Triphosphate

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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