AlanFung:LabNotes/Reprogramming/2011-10-7

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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
  • Run scdna1s
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

Qubit[edit]

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
  • Incubate at 16C for 1 hr

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

PAGE[edit]