3 replies to this topic

[Idit]

Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit

[Clare]

Hi

Do you cool your samples on ice or on a cooly block after denaturation? A few months ago, my labelling stopped working well and I realised my cooly block kinda broke down and wasn't cold enough! Perhaps it's a long shot. but thought it was worth mentioning

Clare

Idit, on Mar 2 2010, 10:31 AM, said:

Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit

[Idit]

Hi Clare,
Yes,  I cool the samples 5 minutes on ice. In what temperature do you do the coupling with the dye?
Thanks,
Idit

Clare, on Mar 3 2010, 11:39 AM, said:

Hi

Do you cool your samples on ice or on a cooly block after denaturation? A few months ago, my labelling stopped working well and I realised my cooly block kinda broke down and wasn't cold enough! Perhaps it's a long shot. but thought it was worth mentioning

Clare

Idit, on Mar 2 2010, 10:31 AM, said:

Hi all,

I'm doing indirect labeling (Aminoallyl + Cy3/5) for a couple of years. I used to get 250-500 pMol of each color with frequency of incorporation (FOI) 10+. Lately I started getting 60-100 pMol with FOI 2-6.

My prorocol is in brief:
1. Amplification with klenow, aminoallyl mix, and random primers ON.
2. The next day I clean with microcon
3. Coupling with the Cy dye in MaHCO3 0.1M buffer for 1.25h. I'm using a fresh buffer each time at pH 8.6.
4. Cleaning with Quiagen purification column.

I tried changing the Aminoallyl stock and the batch of the Cy dye, the klenow enzyme, the random primers. Does anyone have an idea what could be interrupting?

Thanks,
Idit

[Clare]

Hi again

I use a different method (Invitrogen BioPrime kit) but it's 37degC ON.
Clare

[quote name='Idit' date='Mar 3 2010, 12:55 PM' post='61059']
Hi Clare,
Yes,  I cool the samples 5 minutes on ice. In what temperature do you do the coupling with the dye?
Thanks,
Idit