IPCR Protocol

This is a copy of the protocols used to generate the IPCR template for the SINS sequences from the SLAT population - it is also a good guideline for generic IPCR, although obviously the enzymes, temperatures and primers will need to be appropriate for your individual inserts or genes.
  1. Digestion
    DNA 2µl (200ng)
    10x BstYI buffer (NEB) 1µl
    BstYI (10U/µl) 0.2µl
    100x BSA (10 mg/ml) 0.1µl
    dH2O 6.7µl
    10µl

    Digest 1 hour and 30 min at 60°C (can be done in a PCR machine)
    Inactivate the enzyme by heating 20 min at 75°C (can also be done in a PCR machine)

  2. Ligation
    Digested DNA 10µl
    10x ligase buffer 4µl
    Ligase (1 U/µl) 0.2µl
    dH2O 25.8µl
    40µl

    Ligate overnight at 4°C (in a refrigerator)

  3. PCR
    DNA (from ligation) 4.0µl
    10x PCR buffer 2.0µl
    dNTPs (2mM) 2.5µl
    Primer A (10uM) 0.5µl
    Primer B (10uM) 0.5µl
    Taq/Pwo (5U/µl) 0.2µl Ratio Taq/Pwo (Boehringer) of 160/1 unit
    dH2O 10.3µl
    20µl

    Cycling parameters:
    94°C 2min
    94°C
    65°C
    68°C
    		 10 sec
    30 sec
    5 min
    		38 cycles
    (extension is 45 sec to 1 min per kb)
    68°C 5 min
    The PCR machine is preheated to 94°C before inserting the plate containing the PCR mix and DNA.
Buffers:
10x Ligase buffer: 200mM Tris-Cl pH 7.6, 50mM MgCl2, 50mM DTT, 500ug/ml BSA, 5mM ATP

10x PCR buffer: 17.5 mM MgCl2, 500 mM Tris-Cl pH 9.2, 140 mM (NH4)2SO4

Other enzymes used for IPCR (in combination with primers as below):
ApoI digest:
DNA 2µl (200ng)
10x Buffer 3 (NEB) 1µl
ApoI (10U/µl) 0.2µl
100x BSA (10 mg/ml) 0.1µl
dH2O 6.7µl
10µl
Digest 1 hour and 30 min at 50°C (can be done in a PCR machine)
Inactivate the enzyme by heating 20 min at 75°C (can also be done in a PCR machine)

HinDIII digest:
DNA 2µl (200ng)
10x Buffer 2 (NEB) 1µl
HinDIII (10U/µl) 0.2µl
dH2O 6.8µl
10µl
Digest 1 hour and 30 min at 37°C (can be done in a PCR machine)
Inactivate the enzyme by heating 20 min at 75°C (can also be done in a PCR machine)

Ligation and PCR same as for BstYI restricted-ligated DNA

Primers used:

BstYI:
DSPM1 - SPMINV4
DSPM5 - SPMINV3

ApoI:
DSPM1 - SPMINV12
DSPM5 - SPMINV8

HinDIII :
DSPM1 - SPMINV14
DSPM5 - DSPM9

DSPM1: CTT ATT TCA GTA AGA GTG TGG GGT TTT GG
DSPM5: CGG GAT CCG ACA CTC TTT AAT TAA CTG ACA CTC
SPMINV4: CTG GAT CCT GGC ATG ACG TGG GTT TC
SPMINV3: GCG AAT TCA AGT ATG ACG GGC TGA TAC TG
SPMINV12: GTA GAA AGA CAG AGA GCA AGC AAC CAA TG
SPMINV8: CCA CAC GGA CAC TCT TAT GAA TG
DSPM9: GAC AAC ACT GTC CAG CCA AGA C
SPMINV14: GCG AAG TAA TCG CAA CAT CCG CAT TA