The vector pTR-UF3 was digested with pTR-UF3 using AscI and PacI for 5 hours according to the following protocol:
- 10 µg vector
- 2 µL AscI
- 2 µL PacI
- 5 µL 10X BSA
- 5 µL 10X buffer 4
- Up to 50 µL nuclease-free water
21/09/2010
The digested capsid fragments and the vector were loaded on a 1% agarose gel and ran for 50 minutes. The ~2.2 KB (capsid genes) and ~5 KB (vector) fragments were then purified from the gel.
A ligation reaction was carried out according to the following protocol:
- ~3.556 µg insert (Capsid genes)
- ~2.667 µg vector
- 6 µL T4 DNA ligase
- 8 µL T4 DNA ligase buffer
- Up to 80 µL nuclease-free water
- Incubate at 16 ͦC overnight
22/09/2010
The ligation product from the day before was used to transform electrocompetent cells (Invitrogen), according to the following:
- 600 µL of electrocompetent bacteria were mixed with 62.5 µL DNA from the ligation reaction (about 125 ng per 20 µL bacteria), 20 µL of the mixture were placed in the electoporation cuvette
- Electroporation conditions:
- Recovery of bacteria was done by adding 1 mL LB media to each 20 µL of bacteria that were electroporated. The bacteria were collected in a 200 mL flask and incubated at 37ͦC with shaking at 225 rpm for 1 hour.
The bacteria were then used to inoculate 15 cm petri dishes with ampicillin resistance media. 500 µL were spread on each plate, and 50 plates in total were inoculated. The plates were incubated at 37 ͦC overnight.
23/09/2010
The plates from the previous day were collected, and 50 colonies were picked and used to inoculate miniprep cultures.
The colonies from the 50 plates were pooled together and cultured in 700 mL LB for 3 hours, after which a Megaprep (Invitrogen) was done (according to manufacturer's recommendations) to extract the plasmids that contain the shuffled capsid genes and create a library of those shuffled capsids that would be used for the selection of the best capsids, and thus best AAVs.
A left-over from the transormation from previous day was stored in the fridge and used to inoculate three more 15 cm plates, since the other 50 plates were not suitable for counting the colonies (very crowded!) to estimate the size of the library.
24/09/2010
5 flasks (?) of HEK cells were transfected with a total of 75 µg of the library DNA and AAV helper plasmid (1:1) for AAV production, using HBSS. However, the transfection was not successful.
minipreps of the 50 picked colonies were done, and 10 clones were sent for sequencing. Test digestion with AscI, PacI indicated that the clones picked were positive for the insert (capsid genes).
Counting of the colonies on the plates from the previous day showed a library size of (...)?
50 more colonies were picked from one of the plates and used to inoculate mini-prep cultures.
25/09/2010
Analysis of the sequencing results indicated good shuffling of the capsids of two samples, while the 8 others had mainly the sequence of the capsid of AAV5.
50 mini-preps of the colonies picked on the previous day were initiated.
27/09/2010
The 50 samples from the mini-preps from the previous day were test-digested with AscI and PacI, and additionally with NcoI to identify whether AAV5 is present more than the other serotypes. The test digestion with NcoI revealed an AAV5 contamination, so most samples were positive for the AAV5 pattern when digested with NcoI. Sequencing of 10 more samples confirmed this finding.
28/09/2010
The source of the contamination with AAV5 was identified. The second PCR for the shuffled capsids was repeated.
29/09/2010
The 2.2 KB band corresponding to cap genes was purified from the gel that was run for the PCR from the previous day. Digestion of the purified cap genes and the pTR-UF3 was done overnight using AscI and PacI according to the same protocol from the previous time.
30/09/2010
Ligation of the cap genes into the pTR-UF3 vector was carried out accoring to the same protocol from the previous time, the ligation time was 6 hours instead of overnight ligation.
Transformation of Invitrogen electrocompetent cells was done like the previous time and according to manufacturer's recommendations.
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