Results of the first PCR. 8 fragments of each AAV capsid were amplified.
10/09/2010
Results of the second PCR. Fragment 5 from AAV1 as well as fragment 1 from AAV8 were amplified.
13/09/2010
All fragments (F1,F2,F3,F4,F5,F6,F7,F8) from AAV cap1,2,5,6,8,9 were combined. Therefore 8 fragment mixes ready for restriction digest with Bsa1 were obtained. 2ul of PCR amplified fragments were taken for the restriction (NanoDrop analysis showed 1ug of DNA of each of six AAVs giving 6-7ug in total per virobyte).
Bsa1 digestion:
6x2ul of DNA
3ul NEB4 10x buffer
3ul BSA 10x
11ul H2O
=30ul total
digest at 37°C for 1hr
heat inactivation at 65°C for 20min
14/09/2010
All fragments from previous day were washed with QIAquick nucleotide removal kit and the DNA was dissolved in 30ul H2O
Anchor stock solution: Anchor oligos 12689 (150ul) and 5 (30ul) were added to Anchor_comp_all (180ul). The mixture was diluted 10x and then heated to 95°C for 2min and subsequently left to slowly cool down to room temperature on a heating block.
15/09/2010
Streptavidin beads M-280, M-270, MyOne C1 and MyOne T1 activated in two replicates:
wash twice with 75ul of wash/binding buffer and once with 75ul of 1x ligase buffer
20ul of Anchor to the bead solution
incubated at RT for 1hr
magnet applied until the solutions cleared out (M-270 is a bit slower) and the solution discarded
wash twice with 75ul of wash/binding buffer and once with 75ul of 1x ligase buffer
ViroByte addition started with F1, F2, F3 ,F4 and F5 following the standard protocol:
add 20ul of Fragment X
resuspend the beads
add 2.3ul of 10x ligase buffer
add 1ul of T4 ligase
incubate at room temperature for 1hr (shake to avoid beads precipitation)
16/09/2010
last three (F6, F7, F8) fragments added
red replicates washed twice with 75ul W/B buffer and once with 75 ul 1x ligase buffer and diluted to 40ul water
blue replicates washed according to same protocol and diluted into 35ul
PCR amplification with beads:
25ul Phusion 2x mix
20ul of red replicate DNA solution
0.5ul of virbyte_for
0.5ul virbite_rev
4ul water
Cycle = virbyte amp:
30s 94°C
30s 65°C
1m30s 72°C
4°C forever
HindIII digest from beads for blue replicates:
35ul beads solution
4ul NEB2 10x
1ul HindIII
incubate at 37°C for 1hr
inactivate at 65°C for 20min
blue replicate PCR:
25ul Phusion 2x mix
20ul of digest blue replicate DNA solution
0.5ul virbyte_for
0.5ul virbite_rev
4ul water
Cycle - virbyte amp
20/09/2010
Assembly of three fragments under diffrent conditions
sample 1: 100ng of each fragment, gel purified
sample 2: 100ng of each fragment, nucleotide removal purified
sample 3: 200ng of each fragment, nucleotide removal purified
sample 4: 100ng of each fragment, gel purified (duplicate of sample 1)
sample 5: 300ng of each fragment, nucleotide removal purified
PCR protocol:
20 cycles
30sec 95°C
30sec 60°C
1min 72°C (Taq Polymerase)
1 cyle
5min 72°C
4°C
PCR of the samples with 6 sets of primers
a) virobyte_fw + fragment1_cap1_rv
b) virobyte_fw + fragment1_cap2_rv
c) virobyte_fw + fragment1_cap5_rv
d) virobyte_fw + fragment1_cap6_rv
e) virobyte_fw + fragment1_cap8_rv
f) virobyte_fw + fragment1_cap9_rv
PCR products
1 = 1kb ladder
2-7 = sample 1 with primer pairs a, b, c, d, e, f
8-13 = sample 2 with primer pairs a, b, c, d, e, f
14-19 = sample 3 with primer pairs a, b, c, d, e, f
20-25 = sample 4 with primer pairs a, b, c, d, e, f
26 = 1kb ladder
27-32 = sample 5 with primer pairs a, b, c, d, e, f
33 = positive control (fw_aav1_wildtype_fragment1 + rv_aav1_wildtype_fragment3 with aav1 cap gene)
21/09/2010
Assembly of all eight fragments under determined conditions: 200n of DNA and 15min ligation
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
1min 45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
PCR products
1 = 1kb ladder
2 = 8 fragment assembly
3 = duplication
4 = triplicate
22/09/2010
8 fragment ligation under different conditions
sample 1: repeat of the last assembly with 200ng of each fragment and 15min ligation time
sample 2: sinking DNA concentration from 200ng on.
200ng of DNA are added to the ligation reaction for the fragment 1 to 3
150ng of DNA are added to the ligation reaction for the fragment 4 and 5
100ng of DNA are added to the ligation reaction for the fragment 6 and 7
80ng of DNA are added to the ligation reaction for the fragment 8
sample 3: sinking concentration
300ng of DNA of DNA are added to the ligation reaction for the fragment 1 to 3
250ng of DNA are added to the ligation reaction for the fragment 4 and 5
150ng of DNA are added to the ligation reaction for the fragment 6 and 7
100ng of DNA are added to the ligation reaction for the fragment 8
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
1min 45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
PCR products
1 = 1kb ladder
2 = PCR repeat of sample from 21/09/10
3 = sample 1
4 = sample 2
5 = sample 3
6 = sample 1 duplicate
7 = sample 2 duplicate
8 = sample 3 duplicate
9 = positive control: amplification of fragment 1 from AAV1 cap gene
23/09/2010
step to step assembly
sample 1: fragment 1 and 2 are assembled to the anker
sample 2: fragment 1, 2 and 3 are assembled t the anker
sample 3: fragment 1 to 4 are assembled to the anker
200ng of each fragment are used for every sample and 10min of ligation time
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
1 = 1kb ladder
2 = sample 1 pcr amplified with virobyte_fw primer and F2_cap1_rv primer
3 = sample 1 pcr amplified with virobyte_fw primer and F2_cap2_rv primer
4 = sample 1 pcr amplified with virobyte_fw primer and F2_cap5_rv primer
5 = sample 1 pcr amplified with virobyte_fw primer and F2_cap6_rv primer
6 = sample 1 pcr amplified with virobyte_fw primer and F2_cap8_rv primer
7 = sample 1 pcr amplified with virobyte_fw primer and F2_cap9_rv primer
8 = control of sample 1: fragment 1 and 2 amplified from AAV1 cap gene
9 = sample 2 pcr amplified with virobyte_fw primer and F3_cap1_rv primer
10 = sample 2 pcr amplified with virobyte_fw primer and F3_cap2_rv primer
11 = sample 2 pcr amplified with virobyte_fw primer and F3_cap5_rv primer
12 = sample 2 pcr amplified with virobyte_fw primer and F3_cap6_rv primer
13 = sample 2 pcr amplified with virobyte_fw primer and F3_cap8_rv primer
14 = sample 2 pcr amplified with virobyte_fw primer and F3_cap9_rv primer
15 = control of sample 2: fragment 1, 2 and 3 amplified from AAV1 cap gene
16 = sample 3 pcr amplified with virobyte_fw primer and F4_cap1_rv primer
17 = sample 3 pcr amplified with virobyte_fw primer and F4_cap2_rv primer
18 = sample 3 pcr amplified with virobyte_fw primer and F4_cap5_rv primer
19 = sample 3 pcr amplified with virobyte_fw primer and F4_cap6_rv primer
20 = sample 3 pcr amplified with virobyte_fw primer and F4_cap8_rv primer
21 = sample 3 pcr amplified with virobyte_fw primer and F4_cap9_rv primer
22 = control of sample 3: fragment 1 to 4 amplified from AAV1 cap gene
24/09/2010
8 fragment assembly
200ng of each fragment are used for every sample and 10min of ligation time
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
1min 45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
1 = PCR product of the 8 fragment assembly
2 = PCR product of control: AAV1 cap gene amplified from fragment 1 to 8
3 = 1kb ladder (the ladder is not seperated properly in the higher range due to the short time of running)
25/09/2010
After gel purification the fragments were amplified again
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
1min 45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
1 = 1kb ladder
2 = PCR product of purified assembly from fragment 1 to 4 amplified with virobyte_fw primer and F4_cap1_rv primer
3 = PCR product of purified assembly from fragment 1 to 4 amplified with virobyte_fw primer and F4_cap2_rv primer
4 = positive control: fragment 4 amplified from AAV1 cap gene
5 = 1kb ladder
1 = 1kb ladder
2 = PCR product of purified assembly from fragment 1 to 8 amplified with virobyte_fw primer and virobyte_rv primer
3 = duplicate
4 = triplicate
5 = PCR product (from another pcr) of purified assembly from fragment 1 to 8 amplified with virobyte_fw primer and virobyte_rv primer
6 = duplicate
7 = positive control: cap gene (fragment 1 to 8) from AAV1
27/09/2010
amplification of the assembled 8 fragment, which where gel purified
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
1min 45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
PCR products
1 = 1kb ladder
2 = PCR product of the 8 fragment amplification, which where gel purified. 4µl instead of 2µl are used for the PCR.
3 = duplicate
4 = PCR product (of another PCR) of the 8 fragment amplification, which where gel purified. 4µl instead of 2µl are used for the PCR.
5 = positive control: pcr product of AAV1 cap gene from fragment 1 to 8
6 = 1kb ladder
28/09/2010
repeat of the 4 fragment assembly
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 60°C
45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
1 = 1kb ladder
2 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap2_rv primer
3 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap5_rv primer
4 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap6_rv primer
5 = positive control: fragment 1 to 4 amplified from AAV1 cap gene**6 =
6 = 1kb ladder
29/09/2010
repeat of the 4 fragment assembly
PCR protocol
1 cycle
5min 95°C
25 cycles
30sec 95°C
30sec 51°C
45sec 72°C (Phusion Polymerase)
1 cyle
5min 72°C
4°C
The annealing temperature was dropped to 51°C
1 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap1_rv primer
2 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap2_rv primer
3 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap5_rv primer
4 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap6_rv primer
5 = 4 fragment assembly pcr amplified with virobyte_fw primer and F4_cap8_rv primer
6 = positive control: fragment 1 to 4 amplified from AAV1 cap gene
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