Again: FIX II library? More details

[dboehm]

Dear helpful scientist

Thank you Frederik vor help, but I was as careful to order all from
stratagene.

At the moment I could not FIX II up the problem, so I would be very glad
about some more discussion and help.
Therefor I mention more details about my protocol in the hope that
anyone can find the mistake. I send the same details
to stratgene technical service, and I will publich the result here.

I'm working on the construction of a genomic phage library of a mutant
mouse genome, and I ordered the  complete set of FIX-II Partial Fill-in
Kit, Ligation-Kit and Klenow- Fill-in Kit.

So, the yield of clones after packaging is not satisfying ! The Phage
arms disappear after ligation to Sau3AI partial digested (0.1 u/ 2.5
microg DNA)
and filled-in DNA, but I got in the first reaction only 12000 pfu (full
Pack-reaction)
(Packaging control gave 1,25 E9 pfu)

So, the hypothesis arose, that to much genomic DNA (2 micro g in this
ligation) to 1 micro g FIX-II Vector is to much.
Now I ligated 0,4 microg DNA to 1 microg FIX-II in the hope to get more
pfu.

The result was 14000 pfu complete.

At the moment, I have no idea what to do !

I followed Your protocol as mentioned:

1. Check quality of genomic DNA on 0.3 % agarose gel ==> DNA comigrates
with undigested
   lambda DNA without smear in low molecular region
2. Partial digestion of DNA in a range from 0.25 u to 0.0125 per 2.5
microg DNA
   Digestion 15 min. 37 °C
3. Partial digestion of 50 microg genomic DNA scaled at a enzyme
concentration
   of 0.1u/2.5 microg ==> Running 0.6 % Testgel shows a bulk between 15
kb (kb ladder) and
   21 kb (Lamda Hind III)
4. Purification by Phenol / Chloroform Extraction + Chloroform
exctraction
5. EtOH precipitation, disolved 1n TE
6. dGTP, dATP - Partial Fill-in 45 min, RT together with control fill-in

7. Phenol / Chloroform extraction, Chloroform extraction, EtOH
precipitation, disolved
   in 10 microl TE
8. measurement of concentration by UV-Photometer
9. 1 microl on 0.3 % Testgel shows the bulk of genomic DNA after alle
steps of
   Digestion, Fill-in and Purification still between 12 and 20 kb
10.ligation, ON, 4 °C , 0,4 microg of DNA to 1 microg FIX II
11.packaging of 4 microl, 1h 45 min, RT (22 °C)
12.plating and titering the whole library on 10 x 150 mm NZY plates
after 15 min (37 °C)
   incubation of 70 microl extract (+ 500 microl SM-buffer + 20 microl
chloroform, zentr.)
   together with 600 microl fresh prepared plates cells (XLI-Blue MRA
PII)
   (OD = 0,65 in 10 mM MgSO4, grown in LB (10 mM MgSO4/0,2 % Maltose))
13.Incubation ON, 37 °C

My hypothesis now is:
1. the amount of genomic DNA (0.4 microg) in ligation is too high, so
maybe I should
   lower it to 0.2-0.3 (or less ?)
2. 0.1 u Sau3AI/2.5 microg DNA gives too big fragments, which were
ligated, but not packed.



Is my hypothesis right ? The information is important because ligation
and packaging in expensive and time consuming.

I would be very glad about helpful discussion - as soon as possibly

Thank you

Detlef Böhm

Institut for Human Genetics Göttingen
Heinrich-Düker Weg 12
D-37073 Götingen