Description
Annealing Buffer for DNA Oligos (5X) can not only be used for the annealing of conventional DNA oligos, but also for DNA oligos that are difficult to anneal for RNAi (also called siRNA) plasmid construction. DNA oligos used for RNAi plasmid construction usually contains about 20 complementary sequences that are easy to form hairpin structures, thus affecting the correct annealing of DNA oligos. The use of this product can effectively prevent DNA oligos from forming hairpin structures to ensure correct DNA oligo annealing and enable high efficient ligation with digested plasmids subsequently.
This product is easy to use. Users just need to mix DNA oligos with this product in an appropriate ratio, and place the mix in a thermocycler to complete the annealing in about 90 minutes.
This product is sufficient for 50 annealing reactions with a reaction volume of 100μl.
Packing List：
| Item |
Component |
Quantity |
| D0251 |
Annealing Buffer for DNA Oligos (5X) |
1ml |
| Manual |
– |
1 copy |
Storage Conditions：
Store at -20ºC for up to 1 year.
Precautions：
This product is for DNA oligos only, and can not be used for annealing of RNA oligos.
This product is for R&D only. Not for drug, household, or other uses.
For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instruction
Instructions for Use：
1. Dilute the DNA oligo to be annealed to 50µM in sterilized Milli-Q water or ddH2O. Thaw the Annealing Buffer for DNA Oligos (5X) and mix well before use.
2. Set up the reaction by adding the reagents in order as follows and mix well. If the thermocycler used does not have a heated lid, add one drop of mineral oil to prevent evaporation.
| Nuclease-free Water |
40µl |
| Annealing Buffer for DNA Oligos (5X) |
20µl |
| DNA Oligo A (50µM) |
20µl |
| DNA Oligo B (50µM) |
20µl |
| Total Volume |
100µl |
3. Transfer the reaction tube to a thermocycler and perform the annealing condition as follows:
| Step |
Temperature |
Time |
Description |
| 1 |
95ºC |
2 minutes |
Oligo denaturation |
| 2 |
0.1ºC /8s, to 25 ºC |
~ 90 minutes |
Annealing |
| 3 |
4ºC |
∞ |
Storage |
Note 1: Using Bio-Rad’s T100 as an example, set up the program as follows: 1. 95ºC, 2:00; 2. 95º, 0.08, -0.1ºC per cycle; 3. GOTO step 2, 700X; 4. 4ºC, ∞.
Note 2: If the thermocycler available could not reduce the temperature by 0.1ºC, it can also be set to decrease at 1ºC every 90 seconds. Using Bio-Rad’s T100 as an example, set up the program as follows: 1. 95ºC, 2:00; 2. 95ºC, 1:30, -1ºC per cycle; 3. GOTO step 2, 70X; 4. 4ºC, ∞.
Note 3: When a thermocycler is not available, the PCR tube can be placed in a 95ºC water bath that can naturally cool down to 25ºC within 1-2 hours. However, the annealing efficacy will be inferior to that obtained using a thermocycler.
4. The annealed products can be used directly for ligation, or stored at -20ºC for future use. For other applications such as enzyme digestion, the annealed products should be purified with a purification kit, or should not exceed 5% of the reaction volume to avoid the interference of the Annealing Buffer.
References
Related papers：
1. Jiang W, Liang P, Wang B, Fang J, Lang J, Tian G, Jiang J, Zhu TF.
.
Nat Protoc. 2015 May;10(5):768-79. (IF 10.419)
2. Fang H, Dong Y, Yue X, Hu J, Jiang S, Xu H, Wang Y, Su M, Zhang J, Zhang Z, Wang N, Chen X
.
Plant Cell Environ. 2019 Jul 42(7):2090-2104. (IF 6.362)
3. Jiaxuan Chen, Haitao Chen, Haoming Mai, Shuang Lou, Mengqi Luo, Haisheng Xie, Bin Zhou, Jinlin Hou, De-Ke Jiang
.
Hum Mol Genet. 2023 Apr 6;32(8):1334-1347. doi: 10.1093/hmg/ddac284. (IF 5.1)
4. Qu B, Han X, Tang Y, Shen N.
.
PLoS One. 2012;7(7):e41504. (IF 2.74)
5. Huang G, Gao Q, Zhao Y, Dong Z, Li T, Guan X, Jiang J.
.
Gene. 2015 Mar 10;558(2):278-86. (IF 2.984)
6. Yuanyou Li, Ziyan Yin, Yewei Dong, Shuqi Wang, Óscar Monroig, Douglas R Tocher, Cuihong You
.
MAR BIOTECHNOL. 2019 Feb;21(1):19-29. doi: 10.1007/s10126-018-9854-0. (IF 2.798)
7. Tian M, Qu J, Li P, Bao Y, Liu J, Ding C, Wang S, Li T, Qi J, Yu S
.
Vet Microbiol. 2019 Mar 230:130-137. (IF 3.03)
8. Chen H, Wang J, Cui J, Wang C, Liang S, Liu H, Wen J
.
APPL MICROBIOL BIOT. 2019 Aug 103(16):6629-6644. (IF 3.53)
9. Chang Y, Deng Y, Li T, Wang J, Wang T, Tan F, Li X, Tian K
.
Transbound Emerg Dis. 2020 Mar 67(2):564-571. (IF 4.188)
10. Yaokang Wu, Yanfeng Liu, Xueqin Lv, Jianghua Li, Guocheng Du, Long Liu
.
Biotechnol Bioeng. 2020 Jun;117(6):1817-1825. doi: 10.1002/bit.27322. (IF 4.002)
11. Tiyu Ding, Sumathi Tomes, Andrew P Gleave, Hengtao Zhang, Andrew P Dare, Blue Plunkett, Richard V Espley, Zhiwei Luo, Ruiping Zhang, Andrew C Allan, Zhe Zhou, Huan Wang, Mengmeng Wu, Haiqing Dong, Chonghuai Liu, Jihong Liu, Zhenli Yan, Jia-Long Yao
.
Hortic Res. 2022 Jan 18:9:uhab007. doi: 10.1093/hr/uhab007.
12. Xueyang Chen, Xingming Wang, Yuxin Yang, Chun Fang, Jing Liu, Xiongyan Liang, Yuying Yang
.
Front Vet Sci. 2022 Nov 14:9:1031480. doi: 10.3389/fvets.2022.1031480. (IF 2.245)
13. Liu-Qing Yang, Han-Yin Hu, Yao Han, Ze-Yi Tang, Jie Gao, Qi-Yin Zhou, Yi-Xuan Liu, Hao-Sa Chen, Tu-Nan Xu, Lei Ao, Ying Xu, Xuan Che, Ya-Bo Jiang, Chun-Wei Xu, Xian-Chao Zhang, Yu-Xin Jiang, Michal Heger, Xiao-Min Wang, Shu-Qun Cheng, Wei-Wei Pan
.
Cancer Gene Ther. 2022 Dec;29(12):1895-1907. doi: 10.1038/s41417-022-00503-z. (IF 4.534)
14. Jieru Wang, Xiaojie Zhu, Dongdong Yin, Chang Cai, Hailong Liu, Yuqing Yang, Zishi Guo, Lei Yin, Xuehuai Shen, Yin Dai, Xiaocheng Pan
.
Microorganisms. 2023 Jan 31;11(2):354. doi: 10.3390/microorganisms11020354.
15. Minggui Lin, Xingyun Liu, Xinhui Zhang, Huimin Wang, Yu Fang, Xiaoting Wu, Anqi Yin, Wanqing Yang, Dong Zhang, Miaomiao Li, Ling Zhang, Songcheng Ying
.
Immunol Invest. 2023 Feb;52(2):224-240. doi: 10.1080/08820139.2022.2157283. (IF 2.511)
16. Shuhui Zhang, Hui Wang, Tong Wang, Wenjun Liu, Jing Zhang, Hongcheng Fang, Zongying Zhang, Futian Peng, Xuesen Chen, Nan Wang
.
Plant J. 2023 Mar;113(5):1062-1079. doi: 10.1111/tpj.16100.
17. Jie Wu, Hongpan Xu, Fenghua Hu, Yiyue Jiang, Boyue Fan, Adeel Khan, Yifan Sun, Kaili Di, Xinrui Gu, Han Shen, Zhiyang Li
.
Front Bioeng Biotechnol. 2023 May 3:11:1169424. doi: 10.3389/fbioe.2023.1169424.
18. Xihui Wang, Cui Zhao, Xinyao Lu, Hong Zong, Bin Zhuge
.
J Agric Food Chem. 2023 Jun 14;71(23):8981-8990. doi: 10.1021/acs.jafc.3c01722.
19. Yang Li, Hanying Huang, Ziang Zhu, Shuzhao Chen, Yang Liang, Lingling Shu
.
iScience. 2023 Jul 22;26(8):107451. doi: 10.1016/j.isci.2023.107451.
20. Shuhui Zhang, Hui Wang, Tong Wang, Jing Zhang, Wenjun Liu, Hongcheng Fang, Zongying Zhang, Futian Peng, Xuesen Chen, Nan Wang
.
Plant Physiol. 2023 Jul 3;192(3):2081-2101. doi: 10.1093/plphys/kiad119.
21. Yuanyuan Chen, Hui Shen, Tingting Liu, Kun Cao, Zhijie Wan, Zhipeng Du, Hang Wang, Yue Yu, Shengzhe Ma, Edward Lu, Wei Zhang, Jianming Cai, Fu Gao, Yanyong Yang
.
J Exp Clin Cancer Res. 2023 Sep 30;42(1):256. doi: 10.1186/s13046-023-02829-4.
22. Dongdong Yin, Lei Yin, Jieru Wang, Yin Dai, Xuehuai Shen, Ruihong Zhao, Kezong Qi, Xiaocheng Pan
.
Avian Pathol. 2023 Dec;52(6):438-445. doi: 10.1080/03079457.2023.2254253.
23. Demin Du, Yueyan Yang, Yuanyuan Zhang, Guanxiong Wang, Liying Chen, Xiaowei Guan, Lene Juel Rasmussen, Dekang Liu
.
Cell Mol Biol Lett. 2024 Mar 14;29(1):37. doi: 10.1186/s11658-024-00547-z.
24. Tengteng Huang, Xiaoling Chen, Jun He, Ping Zheng, Yuheng Luo, Aimin Wu, Hui Yan, Bing Yu, Daiwen Chen, Zhiqing Huang
.
Elife. 2024 Jun 24:12:RP90724. doi: 10.7554/eLife.90724.
