Amplify POLR1D Gene[edit]
Material[edit]
- Total RNA (1ug/ul) (Clontech-from Rui)
- GSP-Gene specific primer
- 1 step RT-PCR Kit
Primer Design[edit]
- I received this pair of primers from Sergio
CGG(RE)ATGGAAGAGGATCAGGAGCTGGAGAG (Forward) 5’-3’
CGG(RE)CTAGAATGTGGATTCATTTCTGCTTGC (Reverse) 5’-3’
- I am not going to add restriction enzyme, and hence I will just order the primers without the RE sites.
- After blasting the primers o NCBI blast the the primers matched 100% to POLR1D gene when cgg is removed from 5' end
- Order from IDT after confirming with Kun
ATGGAAGAGGATCAGGAGCTGGAGAG (Forward) 5’-3’
CTAGAATGTGGATTCATTTCTGCTTGC (Reverse) 5’-3’
- After blasting the primers aligns at transcript NM_015972.3 this locus is 817bp long, with primer 3 the product size is 402 with Tm at 67.46C and 63.40C (avg~65C)for forward and reverse primer respectively. I will use annealing temperature at 55C
cDNA Synthesis & RT-PCR with Superscript III[edit]
Some parameters to be considered[edit]
- Starting material 10ng total RNA
- Preheat thermal cycler to 45-60C depending on the temperature selected for cDNA synthesis (Protocol recommends 30min incubation at 55C as a starting point)
- For high gc content templates use a 60C cDNA synthesis temperature
- cDNA synthesis can be accomplished in a 15-30min incubation at 45-60C. For small targets and incubation time of 5min may be sufficient
- Annealing temperature should be 10C below the melting temperature of the primers used
- Extension time varies with the size of the amplicon (approximately 1 min per 1kb of amplicon)
- For all targets up to 4.5kb, 2ul of superscript III RT/Platinum Taq Mix is sufficient
Protocol[edit]
- Dilute primers to 10uM (1:10)
- Dilute RNA to 10ng/ul (1:100)
- Preheat thermal cycler to 55C
- Prepare Reaction on ice (Setup two reactions, 1 with 10ng total RNA, one with 1ul ddH2O as negative control)
Content
|
Volume per reaction
|
MM
|
2X reaction mix |
25 |
55
|
Template RNA (10ng/ul) |
1 |
Do not add to MM
|
Sense primer (10uM) |
1 |
2.2
|
Anti-sense primer (10uM) |
1 |
2.2
|
Superscript III RT/Platinum Taq Mix |
2 |
4.4
|
ddH2O |
20 |
44
|
Total |
50 |
110
|
55C 30min
94C 2min
40x
{94C 15s
62C 30s
68C 1min}
68 5min
Purification[edit]
- Perform Qiaquick purification
PAGE Analysis[edit]
File:ZhangLab 2 2011-10-18 11hr 42min.jpg
Perform Cloning and Sanger Sequencing[edit]
Blunting with NEB end repair kit[edit]
- Quantify the product-Qubit 40.4ng/ul
- Starting material should be 1-5ug
- Take of PCR product
Content
|
POLR1D
|
Fragmented DNA |
24.8
|
10X End Repair Reaction Buffer |
10
|
End Repair Enzyme |
5
|
H2O |
60.2
|
Total Volume |
100
|
- Incubate at 20C for 30min
- Minelute with 10ul EB
20.8ng/ul
Ligating into pCR blunt vector[edit]
- Calculate the amount of PCR product needed for ligation
- Talked to Rui and she used 5ng vector, I will try the same protocol as I got too many colonies from the previous cloning, however I will maintain insert to vector ratio at 10:1
- x ng insert = (10*500*5/3500)
- X=7.14ng needed
- Make 5ng/ul vector (Mix 2ul vector with 8ul water)
Content
|
POLR1D
|
PCR Blunt (5ng/ul) |
1
|
Blunt PCR Product |
0.4
|
10X ligation buffer (with ATP) |
1
|
Sterile water |
6.6
|
T4 DNA Ligase |
1
|
Total Volume |
10
|
Heat-shock transfection[edit]
- Equilibrate water bath to 42C.
- Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
- Thaw the TOP10 E.coli competent cell vial on ice and split 25ul to a new 1.5ml tube
- Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocl).
- Set up conpetent cell vials in the foam-board holder(for water bath). Incubate the vials on ice for 30 mins.
- Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
- Add 125ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall).
- Shake the vials horizontally at 37C for 1 hour using vortex in the incubator at the lowest speed.
- During the 1hr incubation.
- After 1hr shacking mixing, plate 50uL from each transformation vial on agar plates (w/ Kanamycine and xgal)
- Incubate plates at 37C for about 20hr.
Preparation for Sanger Sequencing[edit]
PCR amplification and preparation for sanger sequencing[edit]
- Add 12.5ul of 100ul of F and R primer and add 475ul sterile h2o to make 2.5uM primer mix
Content
|
Volume per reaction
|
MM
|
DNA |
Pick one colony per well |
Do not add to MM
|
H2O |
25 |
440
|
M13-21 F+R Primer (2.5uM each) |
8 |
140.8
|
2X Taq MM |
15 |
264
|
Total |
48 |
N/A
|
- Add 48ul MM to each colony
94C 10 min to break up the cells
94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever