8 replies to this topic

[anonymous]

one thing I would like to know is whether your insert is a pcr product or not. if it's a pcr product, some enzymes may not cut well by introducing cut site into your primers with 4-5 extra bases at the 5' end of your primers.

[anonymous]

i try to ligate a 3KB insert fragment into a 12Kb vector, and they are both cut by a SacI RE.First, i cut the insert fragment and vector with SacI, without purify and dephosphorylate the DNA vector after digestion, i do the ligation directly. the concentration and amout i used as follow:

insert fragment:90ng(0.5ul)vector:10ng(7ul)T4 ligase: 0.5ul5X T4 ligase buffer :2ultotal:  10ul

and then i put the ligation mixture in 16oC overnight. But i found that the cloing was failed.(suppose transformation &competent cell is ok)

Do you think is the problem of religation of Vector? or because the vector is too large, should increase amount of insert DNA and decrease amount of vector? could you suggest some improvement in the ligation or DNA preparation? Thank you

[anonymous]

After you dephosphorylate your vector, you need to get rid of the phosphatse before you do the ligation. The books say you can heat kill, I always do both heat kill and phenol extraction to be sure that when I add the insert, it does not get dephosphorylated.  I would also suggest doing the cloning without dephosphorylating.  Sure, you may have to screen a few more clones to find the one with the right insert,but the cloning is more efficient.-Val Thomson

[anonymous]

After you dephosphorylate your vector, you need to get rid of the phosphatse before you do the ligation. The books say you can heat kill, I always do both heat kill and phenol extraction to be sure that when I add the insert, it does not get dephosphorylated.  I would also suggest doing the cloning without dephosphorylating.  Sure, you may have to screen a few more clones to find the one with the right insert,but the cloning is more efficient.-Val Thomson

[anonymous]

at first, i prepare my insert fragment by pcr from the chromosomal DNA, and then i run the gel and cut the right fragment.later on, i purify the DNA from the gel.in order to get a more concentrated insert DNA, i precipitate the DNA with ethanol.Then i cut the insert(5Kb) and vector (14Kb) with the same enzyme since both of them is single cut , it is a same enzyme ligation.Today, i check the concentration of insert and vector, they are 100ng/ul & 25ng/ul respectively. and the ligation mixture should be 10ul in total. i still dunno how much of insert and vector amont should i put in the ligation.

insert: Xvector: Yligase:0.5ul5X buffer:2ulwater:(7.5-x-y)ul

what amount of X and Y you suggest?

[Jack]

1, Purify your insert fragment after you cut it with the Sac I.2, Dephosphorylate the vector and inactivate the enzyme by heat.3, Try different ratio for Insert:Vector.  There was a case that only 9I:1V worked.  You may run a small amount of your ligation product out together with the insert and vector only, and check whether there is any new band(s).  However, sometimes the plsmid itself is not stable and may go through recombination even in ligation especially when your vector backbone is a small one such as pUC and the final construct is large.  Once I even screened ~1000 colonies to get the right one.  Good luck!

[anonymous]

1, Purify your insert fragment after you cut it with the Sac I.2, Dephosphorylate the vector and inactivate the enzyme by heat.3, Try different ratio for Insert:Vector.  There was a case that only 9I:1V worked.  You may run a small amount of your ligation product out together with the insert and vector only, and check whether there is any new band(s).  However, sometimes the plsmid itself is not stable and may go through recombination even in ligation especially when your vector backbone is a small one such as pUC and the final construct is large.  Once I even screened ~1000 colonies to get the right one.  Good luck!

[anonymous]

You need to use 2ul of your 5X ligation buffer in a 10ul reaction to get a final conc. of 1X.

[anonymous]

First tell me what is the source from which u r extracting DNA. if from blood, triton and SDS (both soaps) are used to lyse the cell membrane and nuclear memrane respectively (both act by reducing the surface tension of the walls; hence lysis).Alcohol (100%) is used for precipatation of DNA and 70% for washing the DNA (to remove salts and organic contaminents, if any)