does glycogen inhibit enzyme digestion or T4 ligation? - (Feb/08/2006 )
i know that at the concentration as high as 8ug/ul of glycogen will inhibit enzyme digestion and T4 ligation. does anyone know whether lower concentration of glycogen will inhibit the reaction or not? my ligation could not work. i am worrying about the glycogen interference. my glycogen final concentration is around 1.2ug/ul. thank you very much in advance.
thank you for your reply
Fred_33 -- aren't you confusing glycogen with glycerol? I don't know of any enzymes stored in 50% glycogen. As far as I know glycogen is inert, simply used as a precipitation aid for DNA and RNA. If you are concerned about its effects, you could consider the alternative of linear polyacrylamide.
you're right. Hence i've deleted my post in order not to confuse someone...
sorry for the mistake.
I found <0.1ug/ul of glycogen do not inhibit T4 RNA ligase, but not sure about T4 DNA ligase.
If you feel something in your sample inhibit ligation, you might purify it before ligation, by uisng spin colum for oligo, such as Roche's mimi Quick Spin Oligo Columns or Qiagene oligo purification kit.
by the way, do you use oligo with 5'P? at least one strand of your oligo should have 5'P...
I have make a mistake in ligation with oligos that both bear 5' OH..
Glycogen will not inhibit ligation like yeast tRNA. Does not interfere with A260/280 readings. Glycogen may compete with proteins in nucleic acid:protein interactions. (Aruffo, A. and B. Seed, 1987 Proc. Natl. Acad. Sci. USA 84:8573-8577 and Gaillard, C. and F. Strauss, 1990 Nucl. Acid Res. 18:378.) Glycogen is reported to inhibit the reverse transcription of large templates in a concentration dependent manner. (Baugh, L.R., A.A. Hill, E.L. Brown and C.F. Hunter 2001. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 29:E29) Ambion does not recommend the use of glycogen for samples that will be used in a microarray.
Excessive concentrations of tRNA used as a carrier in ethanol precipitation will inhibit ligation reactions. Slight inhibition of colonies per µg DNA at 500 µg/ml tRNA and significant inhibition at 2500 µg/ml. Glycogen shows no inhibition at 1000 or 5000 µg/ml (my data 1986 p 3209).
If you feel something in your sample inhibit ligation, you might purify it before ligation, by uisng spin colum for oligo, such as Roche's mimi Quick Spin Oligo Columns or Qiagene oligo purification kit.
by the way, do you use oligo with 5'P? at least one strand of your oligo should have 5'P...
I have make a mistake in ligation with oligos that both bear 5' OH..
My oligo is around 40bp. i first digested it with enzymes and then did the ligation ,therefor the oligo contain 5'P. i only dephosphorylated my vector. can 40bp oilgo be purified by colum or purification kit? i worry the recover efficiency .
Based on the manual of these products:
Use QLAquick Necleotide Removal kit (Qiagen) to purify oligo (17 – 40nt) with 60-80% recovery.
While mini Quick Spin Oligo columes (Roche) to purify oligo (>=8nt) with >=80% recovery.
Quick Spin DNA columns (Rocho) still can purify DNA (>=20bp) with >=90% recovery.
These products work well in my work; I’d feel that even the home made G50 column still work for such size fragment.
By the way, I don’t think you need dephosphorylated vector if you ligate such oligo fragment to vector, the number of oligo fragments should be more excess to you vector…
Use QLAquick Necleotide Removal kit (Qiagen) to purify oligo (17 – 40nt) with 60-80% recovery.
While mini Quick Spin Oligo columes (Roche) to purify oligo (>=8nt) with >=80% recovery.
Quick Spin DNA columns (Rocho) still can purify DNA (>=20bp) with >=90% recovery.
These products work well in my work; I’d feel that even the home made G50 column still work for such size fragment.
By the way, I don’t think you need dephosphorylated vector if you ligate such oligo fragment to vector, the number of oligo fragments should be more excess to you vector…
thank you very much for your useful information. i purified my oligo yesterday and this time i used non-dephorsphorylated vector to do the ligation. lets see what will happen this time. by the way. the ratio of my vector and insertion is 1:15-20.
Hi rose
while I am not sure the nature of you work, although I have successed in ligation of 10bp adapter to a vector, I recommand you read posts in follow link: http://www.protocol-online.org/forums/inde...?showtopic=3579
hope you will find more valuble information from it...