TITLE: RNAi Plasmid Construction using pFGC5941
AUTHORS: Yaowu Yuan

[STEPS]
?. [Amplifying insert from cDNA or gDNA using Phusion PCR]
Amplify insert from cDNA or gDNA (if the fragment contains no intron) using Phusion PCR
 Make TWO  reactions of the following in separate tubes:  AB1Amount (µL)Reagent24 µL5x Phusion Buffer30.5 µL10 mM dNTPs40.6 µLDMSO51.0 µLTemplate60.2 μlPhusion enzyme711.0 µLdH2O81.5 µL5 µM Forward Primer91.5 µL5 µM Reverse Primer1020 µLTotal
20 µl
AB1Amount (µL)Reagent24 µL5x Phusion Buffer30.5 µL10 mM dNTPs40.6 µLDMSO51.0 µLTemplate60.2 μlPhusion enzyme711.0 µLdH2O81.5 µL5 µM Forward Primer91.5 µL5 µM Reverse Primer1020 µLTotal
?. [Digestion]
Digest one insert with NcoI/AscI and the other one insert BamHI/XbaI.   AB1Amount (µL)Reagent22.5 µL10x CutSmart Buffer34.5 µLdH2O41.5 µLNcoI51.5 µLAscI615 µLPCR Product725 µLTotal
See step 12 for BamHI/XbaI digestion.
AB1Amount (µL)Reagent22.5 µL10x CutSmart Buffer34.5 µLdH2O41.5 µLNcoI51.5 µLAscI615 µLPCR Product725 µLTotal
?. [First ligation]
First Ligation (Would like insert to vector molar ratio to 2:1 to 6:1)  AB1Amount (µL)Component22 µLLinearized pFGC5941 digested with AscI/NcoI. (~175ng; adjust volume as needed)34 µLInsert (digested with AscI/NcoI) (~15-30ng)42 µLT4 Ligase Buffer51 µLT4 Ligase611 µLdH2O720 µLTotal
AB1Amount (µL)Component22 µLLinearized pFGC5941 digested with AscI/NcoI. (~175ng; adjust volume as needed)34 µLInsert (digested with AscI/NcoI) (~15-30ng)42 µLT4 Ligase Buffer51 µLT4 Ligase611 µLdH2O720 µLTotal
?. [Colony PCR to check for first insert]
Colony PCR to check for first insert
   AB1Amount (µL)Component28.0 µLdH2O31.0 µL10x buffer40.125 µLdNTPs50.5 µLpFGC5941 2372 F60.5 µLpFGC5941 3082 R70.05 µLTaq810 µLTotal
AB1Amount (µL)Component28.0 µLdH2O31.0 µL10x buffer40.125 µLdNTPs50.5 µLpFGC5941 2372 F60.5 µLpFGC5941 3082 R70.05 µLTaq810 µLTotal
?. [Colony PCR to check for first insert]
Run Colony PCR   ABCD1CycleRepeatsTemperatureTime2Cycle 195°C3:003Cycle 232x95°C0:15455°C0:15572°C1:006Cycle 372°C7:007Cycle 412°Cforever
ABCD1CycleRepeatsTemperatureTime2Cycle 195°C3:003Cycle 232x95°C0:15455°C0:15572°C1:006Cycle 372°C7:007Cycle 412°Cforever
?. [Picking Colonies and Plasmid Prep]
Pick two correct colonies and inoculate into .
[LB+Kan broth]
?. [Digestion]
Digest plasmid with BamHI/XbaI
?. [Second Ligation]
(d Ligadtodioo(n  AB1Amount (µL)Component22 µLVector that contains the first insert digested with BamHI/XbaI  (~175ng; adjust volume
based on concentration)34 µLInsert digested with BamHI/XbaI (done in step 3) (want ~15-30 ng)42 µLT4 Ligase Buffer51 µLLigase611 µLdH2O720 µLTotal
AB1Amount (µL)Component22 µLVector that contains the first insert digested with BamHI/XbaI  (~175ng; adjust volume
based on concentration)34 µLInsert digested with BamHI/XbaI (done in step 3) (want ~15-30 ng)42 µLT4 Ligase Buffer51 µLLigase611 µLdH2O720 µLTotal
?. [Second Colony PCR]
Colony PCR to check for second insert.pFGC5941 3930 F & pFGC5941 4430 R
Vector without insert will give a band of 500bp.
?. [Picking Colonies and Plasmid Prep]
Pick two correct colonies and inoculate into  .
[LB+Kan broth]
?. [Checking plasmid for inserts]
Check plasmid for inserts PCR to check for both inserts: 2372F/3082R or RNAi_R (insert specific) 3930F/4430R or RNAi_F (insert specific)
?. [Sequencing to verify]
Use 4 primers: 2372F, 3082R, 3930F, 4430R
In the sequencing reaction, add DMSO to aid in the sequencing across the restriction enzyme digest sites (the chromatogram peaks usually drop off dramatically right after the digest sites; an alternative strategy is to PCR the final plasmid with 2372F&3082R for the left insert and 3930F&4430R for the right insert and then sequence the PCR product).
Primer sequences: pFGC5941_2372F: CTTCATCGAAAGGACAGTAGAApFGC5941_3082R: CCAAACAGGCTCATAGATACT pFGC5941_3930F: TGTACATCAGAATGTTTCTGAC pFGC5941_4430R: CGCTCTATCATAGATGTCGCTA
?. [Transformation]
Transform into agrobacterium for infiltration.
?. [Digestion]
Incubate samples for  at .
?. [Digestion]
Gel purify digests and save the BamHI/XbaI digested insert for the second ligation.
?. [First ligation]
Incubate at  for .
?. [First ligation]
Transform   into E. coli competent cells (homemade) and plate on Kan plates.
10 µl
?. [Amplifying insert from cDNA or gDNA using Phusion PCR]
Run Phusion PCR program:  ABCD1CycleRepeatsTemperatureTime2Cycle 198°C0:303Cycle 2(32x)98°C0:10458°C (or the ideal annealing temperature)0:20572°C0:306Cycle 372°C5:007Cycle 412°Cfor ever
ABCD1CycleRepeatsTemperatureTime2Cycle 198°C0:303Cycle 2(32x)98°C0:10458°C (or the ideal annealing temperature)0:20572°C0:306Cycle 372°C5:007Cycle 412°Cfor ever
?. [Colony PCR to check for first insert]
Circle the biggest colonies on your plate and label them 1-8.
?. [Colony PCR to check for first insert]
Make a replica plate for your colonies.
?. [Colony PCR to check for first insert]
PCR across the first insert using primers on the vector to check for an insert: An empty vector will give a band of 700bp
?. [Picking Colonies and Plasmid Prep]
Incubate in  shaker overnight .
?. [Picking Colonies and Plasmid Prep]
The next day, do a plasmid prep (mini-prep kit) with 1 of the colonies that grew well.
?. [Digestion]
Incubate at for .
?. [Digestion]
Gel purify digest.
?. [Second Ligation]
Incubate for  at  .
?. [Second Ligation]
Transform  into E.Coli competent cells (homemade) and plate on Kan plates.
10 µl
?. [Picking Colonies and Plasmid Prep]
Incubate in  shaker overnight.
?. [Picking Colonies and Plasmid Prep]
Plasmid prep (mini-prep kit).
?. [Digest Plasmid with BamHI/Xbal]
AB1AmountComponent25 μl 10x CutSmart Buffer312 μl dH2O41.5 μl XbaI51.5 μlBamHI630 μl Plasmid*750 μl Total
AB1AmountComponent25 μl 10x CutSmart Buffer312 μl dH2O41.5 μl XbaI51.5 μlBamHI630 μl Plasmid*750 μl Total
?. [Digest Plasmid with BamHI/Xbal]
for .
?. [Digest Plasmid with BamHI/Xbal]
Gel purify digest.
?. [Ligation #2]
vector that contains the first insert, digested with BamHI/XbaI (~175 ng; adjust volume based on concentration).
   AB1AmountComponent24 μlinsert digested with BamHI/XbaI (done in step 3) (want ~15-30 ng)32 μlT4 ligase buffer41 μlT4 ligase511 μldH2O620 μlTotal
2 µl
AB1AmountComponent24 μlinsert digested with BamHI/XbaI (done in step 3) (want ~15-30 ng)32 μlT4 ligase buffer41 μlT4 ligase511 μldH2O620 μlTotal
?. [Ligation #2]
Incubate for  at .
?. [Ligation #2]
Transform  into E. coli competent cells (homemade) and plate on Kan plates.
10 µl
?. [Colony PCR to check for second insert]
pFGC5941 3930 F & pFGC5941 4430 R Vector without insert will give a band of 500bp
?. [Colony PCR to check for second insert]
Pick two correct colonies and inoculate into.
[LB+Kan broth]
?. [Colony PCR to check for second insert]
Incubate in  shaker overnight.
?. [Colony PCR to check for second insert]
Plasmid prep (mini-prep kit)
?. [Check plasmid for inserts]
PCR to check for both inserts:2372F/3082R or RNAi_R (insert specific) 3930F/4430R or RNAi_F (insert specific)
?. [Sequence to verify]
Use 4 primers: 
2372F, 3082R, 3930F, 4430R
Note: in the sequencing reaction, add DMSO to aid in the sequencing across the restriction enzyme digest sites (the chromatogram peaks usually drop off dramatically right after the digest sites; an alternative strategy is to PCR the final plasmid with 2372F&3082R for the left insert and 3930F&4430R for the right insert and then sequence the PCR product)
?. [Sequence to verify]
Transform into agrobacterium for infiltration.