ligation and transformation problem
Posted 03 July 2011 - 07:35 PM
i'm currenly working with urease operon from Helicobacter pylori and size of urease operon around 6.2kb. i try to ligate into pgem-t, puc18 and now into pbr322, but still can't get the clone. if i use puc18 or pbr322, i get all false positive (white colonies) but if i use pgemt, all blue colonies.i try to screening with digestion (EcoRI)for 2-3hours,i will get the vector size only.
3ul purify pcr product
1ul t4 ligase
5ul 2x ligase buffer
total volume: 10ul, 4C, overnight
3-4ul ligation product
50ul JM109 commercial
basic transform,shake 180 rpm, 37C, 1.5 hours.
I digest pBR322 or pUC18 with EcoRV, then do the t-tailing (70C, 20-30min), then ligate, tranform but i still can't get the clone. please help me to solve this problem or give me some idea or suggestion.
Posted 14 July 2011 - 07:41 PM
The ligation vector:insert is not based on volume, but on molarity.
I think it is better to dephosphorylate your vector also.
Edited by badguy, 14 July 2011 - 07:42 PM.