Navigation Links
A simple method to sequence from bacterial colonies using [a-33P] radiolabeled ddNTPs and Thermo Sequenase

Introduction

In traditional DNA sequencing systems, sequencing products are radioactively labeled using either endlabeled primers or by the incorporation of radiolabeled dNTPs during primer extension. Successful sequencing results produced by these two labeling methods are highly dependent on template quality. DNA containing nicks, breaks, impurities or high secondary structure can cause DNA polymerases to dissociate from the primertemplate complex before specific ddNTP incorporation occurs. This non-specific termination produces bands across all four lanes (BAFLs) or background artifacts, usually making sequence interpretation difficult. These artifacts can be avoided by the use of [(a-33P] labeled ddNTPs. Labeled terminators prevent the visualization of sequencing artifacts because only specifically-terminated primer extension products are labeled.

The Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit(l) employs a one-step cycle sequencing method that allows the use of as little as 20 fmoles of template. Such high sensitivity allows sequencing of plasmid DNA directly from bacterial colonies with minimal purification. This is extremely useful when rapid screening of many different clones is required. The use of Thermo Sequenase and 33P-labeled ddNTPs, combined with the colony preparation outlined below(2), allows for direct colony sequencing with superior results.

Method

1. Select an appropriate sized colony (~2mm in diameter) on the agar plate and aseptically transfer the entire colony to 10ml of delonized water in a sterile 0.5ml tube.

2. Carefully swirl the colony in water with a sterile 200ml pipet tip. Pipet up and down several times until the colony is fully resuspended.

3. Heat the cell suspension at 95C for 5 minutes, then transfer the tube to an ice bath for 5 minutes.

4. Spin down the cell debris at ~10,000rpm in a microcentrifuge for 3 minutes.

5. Remove the entire supernatant (~10ml) and sequence according to the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Protocol (79750)(3).

Results

Sequencing of plasmids in E. coli colonies using the Thermo Sequenase Radiolabeled Terminator Cylce Sequencing Kit.

Discussion

The rapid preparation of bacterial DNA from colonies described above, in combination with the Thermo Sequenase radiolabeled terminator kit, offers an excellent method to sequence plasmid DNA directly from colonies. Sequence information with virtually no artifacts or background is routinely generated and artifacts associated with template purity or premature termination are not seen at all. Gel compression artifacts, caused by G-C base-pairing of sequencing products, can also be eliminated using the dITP reaction mixture provided with the kit.

The results obtained using these protocols are comparable, or even superior to, those generated using template preparations of high quality and purity. It is important to note that the signal intensity of sequence is related to the size of the colony and the copy number of the plasmid. Therefore, colonies to be sequenced should be at least 2mm in diameter. If the size of the colony is smaller than 2mm or if plasmid is present in low copies, increasing the number of cycles should be beneficial. There is no difference in the quality of sequence generated from fresh or older colonies.

Note: The simple method of colony treatment before sequencing with the Thermo Sequenase radiolabeled terminator cycle sequencing kit was generously offered by Dr. R.S. Ranu at Colorado State University.

References

1. Life Science News , 20, Amersharn Life Science Inc., pp. 2-4. 2.

2. Fan, J., Ranu, R.S. et al., BioTechniques , 21, (1996), pp. 1132-1137.

3. Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit protocol booklet, 79750, USB Corporation.

Related products

Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit, 79750* in USA.

Redivue 33P-labeled terminator pack, AH 9539* from Amersham Pharmacia Biotech

* In the US, the order code is 188403 which includes both 79750 and AH9539.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. New gravity-flow column gives faster and simpler purification of histidine-tagged proteins
2. A simple and rapid LC-MS/MS method for the simultaneous determination of nine antiretroviral drugs commonly used in Europe (protease Inhibitors and non-nucleoside reverse transcriptase inhibitors)
3. Comparison of different protein quantitation methods
4. Advancing the quality control methodology to assess isolated total RNA and generated fragmented cRNA
5. Sensitive detection of tumor cells in peripheral blood of carcinoma patients by a reverse transcription PCR method
6. Outstanding transfection rates using the Multiporator
7. Outstanding transfection rates using the Multiporator
8. Isolation of Cosmids using Eppendorfs Perfectprep Plasmid Mini Kit
9. Normalizing cDNA libraries using the EppendorfThermomixer
10. Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler
11. Efficient DNA transfection of primary CNS neurons using TransMessenger Transfection Reagent
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/26/2016)... ... May 26, 2016 , ... FireflySci has been manufacturing quartz ... all over the globe. Their cute firefly logo has been spreading to more ... makes spectrophotometer calibration standards that never require recalibration. These revolutionary standards have ...
(Date:5/25/2016)... ... 25, 2016 , ... Thailand’s Board of Investment’s New York ... Francisco. Located at booth number 7301, representatives from the Thai Government, research organizations, ... Thai biotechnology and life sciences sector. , Deputy Secretary General of the ...
(Date:5/25/2016)... ... May 25, 2016 , ... Founder ... double board-certified in surgery and surgery of the hand by the National Board ... stranger to going above and beyond in his pursuit of providing the most ...
(Date:5/25/2016)... ... May 25, 2016 , ... Scientists at the University of Athens say they ... may be hampering the research that could lead to one good one. Surviving Mesothelioma ... it now. , The team evaluated 98 mesothelioma patients who got ...
Breaking Biology Technology:
(Date:4/19/2016)... -- The new GEZE SecuLogic access control ... system solution for all door components. It can be ... interface with integration authorization management system, and thus fulfills ... dimensions of the access control and the optimum integration ... considerable freedom of design with regard to the doors. ...
(Date:4/15/2016)... Research and Markets has announced ... 2016-2020,"  report to their offering.  , ... global gait biometrics market is expected to grow ... 2016-2020. Gait analysis generates multiple variables ... to compute factors that are not or cannot ...
(Date:4/13/2016)... physicians supporting Medicaid patients in Central Florida ... telehealth thanks to a new partnership with higi.   ... can routinely track key health measurements, such as blood ... they opt in, share them with IMPOWER clinicians through ... location at no cost. By leveraging this data, IMPOWER ...
Breaking Biology News(10 mins):