Troubleshooting Guide for Plasmid Subcloning

Troubleshooting Guide for Plasmid Subcloning

This cloning troubleshooting guide troubleshoots following aspects of plasmid subcloning:

1. Few or no colonies on control or test plate.
2. Low colony number and 6 or more mini preps are negative.
3. High numbers of colonies on both the Test and the Control plates (i. e. usually 100 or more on the control plate unless cells are very competent).

(eZcloneSystems)

Last update 04-Apr-2003, Rating Fair of 2 votes.

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By richa on 15-Jul-2008

I'm doing the cloning exp for the first time, and my white colonies are not showing my band of interest after the RE digestion. I checked the plasmid on Agarose gel, which was good, but I can see only single band on the gel which is apparently the size of my plasmid. PLasmid size is 3kb and insert size is 1.2 kb, but I can see only 3kb band. What should I do to rectify it?
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By sunil on 09-Jul-2007

i have been cloning the competetent cells (JM101 E.coli) with restricted digested, gel purifed pPICZALPHA A with two gel purified restricted digested PCR primers(Xba1 and EcoR1) in 1:1, 1:2,1:3,2:1,2:0.5. vector to insert ratio. incubated at different temperatures, 15 c, 14 c, 37 c, 4 c.but i havent got any colonies on zeocin plates..all ligation reactions were failure..can any body suggest me how to over come this trouble shooting wih cloning of this 3.6 kb pichia plasmid into E.coli competent cells.
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By NADEEM AHMAD PhD on 28-Sep-2003

heat your insert and vector for 5 minute at 65 c .this will open all insert insert and vector vector ligation then procede for ligation .add ligase at end please tell me after this experiment
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By kaneja on 04-Apr-2003

I am subcloning a 2.5 kb fragment in a plasmid of 5.7 kb. I want to know which molar ration should I try.

I did this experiment 5 times, did not work.
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Troubleshooting Guide for Plasmid Subcloning