A gene is not inserted into the Plasmid - (Feb/09/2011 )

The previous cloned, I've no difficulty and successfully transformed the genes into Dh5 alfa but now, i've a problem to insert another gene (~ 400bp) into Pyes-CBX (~9kb) and transform into yeast S. cerevisiae. I'm not sure either my steps are missing somewhere or I did the wrong things. So I need someone to verify if my steps are wrong

first, try to get the interest gene of the white rot-fungi(~ 400bp) already inside pET3d
2. Run the PCR with primers
3. cut the fragment about 400bp
4. purify with miniprep
5. digest for an hours
6. purify again
7. Transform into yeast
i. 25ml YPD media for yeast cell culture for O/Night
ii. 50ml YPD for the yeast cell culture for 4-5 hours until the OD bet. 0.6 and 1.
iii. Method of AcLi/TE
iv. plate on selective media for 3 to 4 days at 30°C
vi. The colonies are exist on the plate (about 5 to 8 colonies)
vii. HOW TO CHECK THE POSITIVE COLONIES?

8. O/N culture for each colonies in YPD liquid media
i. plus a selective media plate and YPD plate and incubate at 30°C for 3 days
9. Purify O/N YPD liquid media to get a plasmid
10. Digest plasmid
11. Check the gene inside the plasmid through agarose

* I COULD'T SEE THE GENE INSIDE THE PLASMID BUT VERY LOW SIGNAL FOR THE SIZE OF THE PLASMID (~9KB)

the questions
1. How to check the positive colonies
2. Any missing steps?
3. I am not sure for the transformation methods because i did with several protocols but still not working well. Any suggestions?
4. my collegue told me no need to do the ligation, I think something wrong. Do I am right?
5. Honestly, this is my first time to isolated the yeast plasmid with bacteria miniprep kit. However, i used isolation of plasmid DNA from yeast protocol and used acid-washed glass beads (Sigma G-8772). Any suggestions?

I hope someone could help me. Thanks in advance.

Drew

You can check the colonies by PCR, just take the colony with a tip, put it in another plate (for replicate the colony) and then shake the tip inside a pcr tube with your pcr mix (it works, trust me).
And I don't know why you are not doing the ligation... you should try it, something is wrong. After the digestion, you should purify (running a gel) your band, measure the concentration and then set the ligation. After que ligation, do the transformation. It should work...

drew on Wed Feb 9 14:29:36 2011 said:

Thanks Carmela,

Will do it again for the steps of digestion and ligation .. I hope it works ...