PUAST sub - cloning problem - (May/25/2012 )

Dear All

I am trying to insert a 1kb fragment into the vector PUAST (~9kb). Unfortunately, I failed three times in a row but have no clue where did I go wrong. Please give me some suggestions if you have worked /are working on similar cases.

My protocol is the following:

I started with extracting my insert & vector via mini prep. The concetrations for both are roughly 300-400ng/ul

Then I did a digestion for both vector and insert ( vector- 1ug, insert - 5ug) with the restriction enzyme EcROI and NotI, Buffer 3, 2Hrs at 37oC. Heat Inactivate for 15 minutes at 80oC

After that I did a gel extraction for the insert and also gel purifiied it straight after.

For the ligation, I used 2ul vector, 1ul ligase, 4ul ligate buffer and 13ul insert, 15oC overnight

After the transformation, I did a screening by picking 40 colonies and none of them showed a positive result.

The second time, my boss suggested to use more amount of DNA for the insert, so I doubled it to 10ug and other conditions kept the same. However, negatiive results again

The third time, I did not do the gel extraction for the insert as I heard the gel extraction may affect the ligation process. Instead, I did a double digestion with ECoRI and NotI for the first digestion and NCOI for the second. Other conditions were unchanged. failed again.

I am trying very hard on this clone but really have no idea what should I do next. It will be great if somebody can help.
Thank you in advance.

Vincent

the restriction enzyme should be EcoRI. Sorry about that

Vincent,

So you work with flies!

thought to just give you a suggestion protocol:

start with 3 ug of DNA of vector plasmid and 4.5 ug of insert plasmid. Digest each with EcoRI and NotI using NEB buff3 in ~100-150 ul total volume (with a bit of volume you dilute out possible impurities in the mini prep) use 2.5 ul of each enzyme (~10x overdigetion)
After 1-1.5 hours add CIP (calf intestine alkaline phosphatase) to the vector tube only (~2units) mix well and incubate both tubes for 30-45 minutes longer (also at 37C). This will reduce the background (also when using 2 different enzymes). Add gel loading buffer and load the two samples directly on a gel using multiple slots for each sample -because of the volume and to prevent overloading of the gel (for 9 kb use a gel below 1% agarose). Cut out the vector and insert bands carefully (if it is still overloaded just take the front of the band, also, use a long wave length UV box eg 360nm -DNA irradiated with short wave length UV will not clone).
Isolate the DNA from the gel slices and resuspend/collect in a small volume.
Ligate using 1.5 ul of each (will be appr. 1:3 molar ratio) in a total volume of 10ul -using 1 ul of 10x lig buffer 5.25 h2o and 0.75 T4 ligase. ligate o/n at 16C. Also take along controls: one using 1.5 h20 instead of vector and one using 1.5 h2o instead of insert.
Transform the most competent bacteria you have (2.5 ul lig in 50ul cells) and plate 2 dilutions of each transformation (to ensure single colonies on at least one plate).
Determine if the ligation shows enrichment as compared to the control plates.
If it looks good: Pick ~6 free colonies (pick colonies of all sizes present on the plate)
analyze and celebrate (positive thinking helps too!)

Thank you very much. I defintely will try your protocol today. Hope will give you some good news soon