PROTOCOLS!!!

The sequences flanking the mapped Ac elements were cloned using one of the following three inverse PCR protocols after fragment isolation and intramolecular ligation steps needed prior to inverse PCR:

1. iPCR-1 (standard inverse PCR)
2. iPCR-2 (touchdown inverse PCR)
3. iPCR-3 (long range inverse PCR)

or with the AIMS (Amplification of Insertion Mutagenised Sites) protocol.

Please Note: iPCR-2 and iPCR-3 are currently the most efficient protocols for cloning sequences flanking Ac elements up to 8 kb in size (so far...).

Back to projects /// Back to Ac mutagenesis /// Back to Ac lines

Inverse PCR: preparation of DNA samples

Isolation of fragment and intramolecular ligation

•  Digest genomic DNA with the appropriate restriction enzyme in a large volume, using approximately 15 ug DNA in a 200 ul digestion reaction:

Template DNA            ~15 ug
10X Buffer                     20 ul  
BSA                                 2 ul
RNAse                             2 ul  
Restriction Enzyme        10 ul
Water       to volume of 200 ul  

            Incubate at 37 ° for 5 hours.

•  Save ~12 to 20 ul of each digestion (optional: for use in DNA blot analysis in Step 4), and load the remainder into an 0.8% agarose gel, with appropriate DNA markers.   Run overnight.

•  Gel extraction:   Extract appropriate size band based on size estimate.   Follow the Gene Clean III kit (QBIOgene) protocol for gel extraction.   Elute final product in 40 ul.   Run out ~4 ul of each product on a 1% agarose gel for size confirmation.

•  Optional:   To confirm that the correct band was excised, run another southern blot using and Ac-specific probe with 8 to 10 ul of the Gene Clean dilution with the remaining 12 to 20 ul of initial digest Perform DNA blot analysis using Ac -specific probe.

Intra-molecular ligation

•  Use ~20 ng gel extracted DNA (typically 4 to 8 ul) for each ligation and set up 50 ul self-ligation reactions.

20 ng DNA (~4 to 8 ul)
5 ul 10x Ligation Buffer
1 ul Promega T4 DNA Ligase
bring volume up to 50 ul with H20

Run intra-molecular ligation overnight at 15 °C; or at 4 °C for ~36 to 48 hours.   Heat kill enzyme by placing ligation reaction at 70 °C for 10 min. Use QIAGEN Nucleotide removal kit to clean reaction; elute in 50 ul Elution Buffer or H20.

Back to top

iPCR-1: standard
(for amplification of sequences < ~2 kb)

1a. First round PCR reaction mix:

                                                            Volume (ul)/rxn
DNA (ligation product template)                  10 ul
10X buffer (with15mM MgCl2; Promega)     5 ul
DMSO                                                            2 ul
dNTP's (2 mM)                               5 ul
Primer 1 (10 uM)                                          2.5 ul
Primer 2 (10 uM)                                          2.5 ul
Taq polymerase (5U/ul; Promega)               2.5 ul
H2O                                                    up to 50 ul

1b. First round PCR:

1 cycle:    T=94 °C for 2'00"
25 cycles: T=94 °C for 0'45"
                 T=57 °C for 1'00"
                 T=72 °C for 2'00" (1' per kb)
1cycle:      T=72°C for 10'00"
                  T=4 °C hold

2. Second round (nested) PCR:  Add 1 ul of PCR product to 200 ul dH20 and set up 2nd round PCR with nested primers (below) using above PCR reaction mix and cycling conditions.

•  Run out 10 ul of PCR products to identify and confirm sizes of bands. Gel purify and clone fragments.

Back to top

iPCR-2: Touchdown inverse PCR
(for amplification of sequences < ~4 kb)

If the desired amplified sequence is smaller than 5 kb, a touchdown inverse PCR reaction with a regular Taq polymerase can be used to amplify the fragment in a first round of PCR, followed by a second, nested PCR reaction.

1a. First round PCR reaction mix:

•  Add 450 ul water to DNA ligation product elution for a final volume of 500 ul.
•  Use the following reagents for the first inverse PCR round for a final volume of 50 ul:

                                               Volume (ul)/rxn
DNA (ligation product template)     10 ul
10X Buffer (with 15mMMgCl2 )      5 ul
DMSO                                               2 ul
dNTP's (2.5 mM)                               4 ul
Primer 1   (10uM)                               1 ul
Primer 2 (10uM)                                 1 ul
Taq Polymerase (5U/ul)                   0.5 ul
H20                                          up to 50 ul

Forward and Reverse primers used in first round of iPCR, eg. Primers for EcoRI-based Ac flanking sequences:

1b.  First round touchdown PCR protocol:

1 cycle:                T=96 °C for 3'00"
15 cycles:            T=94 °C for 0'45"
                            T=72 °C for 1'00"
                                              -1 °C/cycle
                            T=72 °C for 3'00" (assume 1' per kb)
16 cycles:            T=94 °C for 0'45"
                            T=58 °C for 1'00"
                            T=72 °C for 3'00"
1 cycle:                T=72 °C for 10'00"
                            T=4 °C hold

2a. Second round (nested) PCR reaction mix:

•  Set up the PCR reaction using the same PCR reagent mix as the first round, with the exception of using 10 ul of a 1:50 dilution of first round product with H20 as template DNA and the following nested primers for EcoRI based Ac flanking sequences:

•  Check for amplification products on an agarose gel.  Gel extract and clone the desired product using your favorite cloning procedure.

Back to top

iPCR-3: Long-distance inverse PCR protocol
(for fragments up to ~8 kb in size)

The long-distance PCR can be used for both smaller fragments (~ 2 kb), and fragments up to at least ~8 kb in size.   An extension time gradient is used in the second cycle set in the PCR program.   A Platinum High Fidelity Taq Polymerase (Invitrogen; contains a proof-reading enzyme) is used amplify the products.   Also, the ligated template DNA is not diluted, but used directly from the 50 ul volume of purified ligated template DNA.

1a. Use the following reagents for the long-distance inverse PCR protocol for a final volume of 20 ul:

                                                                      Volume (ul)/rxn
DNA template (~ 3ng)                                             8 ul
10X Buffer                                                               2 ul
MgSO 4 (50 mM)                                                  0.8 ul
Beteine (5 M)                                                            2 ul
DMSO                                                                   0.8 ul
dNTPs (5 mM)                                                       1.2 ul
Primer 1   (10 m M)                                                0.6 ul
Primer 2 (10 m M)                                                  0.6 ul
Platinum High Fidelity Taq Polymerase (5U/ul)     0.1 ul
H20                                                                         3.9 ul

The primers used for amplification of Eco R1-based, Ac flanking sequence are the same as those used in the first round of touchdown inverse PCR.   Template DNA in the final 20 m l volume can range fairly wildly from ~3 to ~20 ng; or up to 60 ng)

The following PCR protocol is used for the long-distance PCR reaction:

1 cycle:               T=94 °C for 4'00"
10 cycles:            T=94 °C for 0'10"
                            T=58 °C for 1'00"
                            T=68 °C for 9'00" (use appropriate extension time; assume 1' per kb)
25 cycles:            T=94 °C for 0'10"
                            T=58 °C for 1'00"
                            T=68 °C for 9'00"
                                            +0'10"/cycle
1 cycle:                T=72 °C for 20'00"
                            T=4 °C hold

Back to top

Ac-AIMS (Amplification of Insertion Mutagenised Sites)

Digestion:

for each sample you need ~200-300 ng of good quality gDNA

gDNA                                           x ul
BSA (100X) if needed               0.1 ul
enzyme specific buffer (10X)        1 ul
Restrictin enzyme                          5 U
Water                                  up to 10 ul

digest for 1 hour at 37 °C (according to enzyme)

Ligation:

Prepare adaptor: mix 50ul of filament 1 [100um] with 50ul of filament 2 [100um], denature for 2 minutes at 92 °C, renature at 65 °C for 10 minutes and then let sit at room temperature for at least 15 minutes. Store at -20 °C.

For each sample, add to the above digestion mixture:

10X ligation buffer (usually use NEB)      2.5 ul
DNA ligase                                                   1 ul
Adapter (50 m M)                                         1 ul

water up to 25 ul (15 ul of ligation + 10 ul from the digestion)

Room temperature for 1 hr, then 37 °C for 2 hours. Go to the next step immediately!

Linear amplification with biotinilated Ac primer:

To the above 25 ul of ligation mixture, add 75 ul of the following reaction mixture:

Ac5 Bio primer (100 uM)                                        0.6 ul
dNTPs (25 mM each, Roche)                                  1.4 ul
10X buffer system (Roche)                                       10 ul
Expand Long Taq mixture (3.5 U/ ul) (Roche)      0.75 ul
H2O                                                                     62.25 ul
    

PCR conditions:

1 cycle:          T=94 °C for 2'00"
12 cycles:      T=94 °C for 0'10"
                      T=64 °C for 0'30"
                      T=68 °C for 2'00"
1 cycle:          T=68 °C for 3'00"
                       T=4 °C hold

Step Two: Cleaning of PCR products

Use the Qiagene PCR product purification and follow the protocol provided. Elute DNA in 60 ul of water (expect to recover 50 ul).

Streptavidin binding:

Mix the beads (Dynabeads (R)M-280 Streptavidin) in the bottle well, use 5-10 ul for each sample. Wash with 100 ul of 1X washing buffer 3 times. Re-suspend in 50 ul of 2X washing buffer, add 50 ul of the eluted DNA. Mix well and keep at room temperature for 15 min or longer (can store sample at 4 °C overnight).

Put the plate on a 96-well magnetic rack, wait for 1 or more min. Remove the aqueous phase from the wells. Add 100 ul of 1X washing buffer and switch positions of the plate on the magnetic rack for 5 times. Remove the washing buffer. Repeat this washing step 3 times. Wash the beads with 100 ul of water as the last washing step. Re-suspend the beads in 50 ul of TE (10 mM Tris pH 8.0; 1 mM EDTA). Store at 4 °C.

Step Three: Exponential amplification (Roche Expand L. PCR System)

For each sample:

DNA on beads (mix the beads well)                            1 ul
dNTPs (25 mM each, Roche)                                     0.7 ul
Ac5 Nest (or Ac3 Nest)(100 uM)                             0.25 ul
Selectable primer (100 uM)                                      0.25 ul
10X buffer system1 (Roche)                                         5 ul
Expand Long TAQ mixture (3.5 U/ ul) (Roche)      0.75 ul

Water up to 50 ul final volume

PCR conditions:            

1 cycle:          T=94 °C for 2'00"
35 cycles:       T=94 °C for 0'10"
                      T=50 °C for 0'30"
                      T=68 °C for 2'00"
                      T= 68 °C for 5'00"
                      T= 4 °C hold

2X concentrated W&B Buffer:

                                    10 mM Tris-HCl (pH 7.5)
                                    1 mM EDTA
                                    2 M NaCl (final concentration 1 M)

Table 1: Biotinilated Ac Primers

Table 2: Adapters and Selectable Primers

Table 3: Ac primer for nested PCR

Back to top

Back to projects /// Back to Ac mutagenesis /// Back to Ac lines