pGEM - T e c h n i c a l M a n u a l pGEM ®-T and pGEM...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: T e c h n i c a l M a n u a l pGEM ®-T and pGEM ®-T Easy Vector Systems INSTRUCTIONS FOR USE OF PRODUCTS A1360, A1380, A3600 AND A3610. PRINTED IN USA. Revised 12/10 Part# TM042 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM042 Revised 12/10 Page 1 1. Introduction ........................................................................................................2 A. Vector Features ....................................................................................................2 B. Important Considerations for Successful T-Vector Cloning..........................2 2. Product Components and Storage Conditions ............................................3 3. Protocol for Ligations Using the pGEM ®-T and pGEM ®-T Easy Vectors and the 2X Rapid Ligation Buffer .......................4 A. Ligation Protocol .................................................................................................4 B. Optimizing Insert:Vector Molar Ratio ..............................................................5 4. Transformations Using the pGEM ®-T and pGEM ®-T Easy Vector Ligation Reactions ...........................................6 A. Transformation Protocol ....................................................................................6 B. Example of Transformation Efficiency Calculation ........................................7 C. Screening Transformants for Inserts .................................................................8 5. pGEM ®-T and pGEM ®-T Easy Vector Sequences, Multi-Cloning Sites and Circle Maps ...........................................................8 A. Sequence and Multi-Cloning Site of the pGEM ®-T Vector ...........................8 B. pGEM ®-T Vector Map and Sequence Reference Points .................................9 C. Sequence and Multi-Cloning Site of the pGEM ®-T Easy Vector ...............10 D. pGEM ®-T Easy Vector Map and Sequence Reference Points......................11 6. General Considerations for PCR Cloning .................................................12 A. PCR Product Purity............................................................................................12 B. Properties of Various Thermostable Polymerases ........................................12 C. Cloning Blunt-Ended PCR Products ...............................................................13 7. Experimental Controls ....................................................................................15 8. Troubleshooting ...............................................................................................16 9. References .........................................................................................................20 10. Appendix ..........................................................................................................20 A. pGEM ®-T Vector Restriction Enzyme Sites....................................................20-T Vector Restriction Enzyme Sites....
View Full Document

This note was uploaded on 10/03/2011 for the course CHEM 113A taught by Professor Professornotknown during the Spring '09 term at San Jose State University .

Page1 / 28

pGEM - T e c h n i c a l M a n u a l pGEM ®-T and pGEM...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online