Chris:LabNotes/FateMapping/Calendar/2016/2016-5-18

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Phi29 Slippage Rate Experiment - Method Comparison

Background

Phi29 Error "PCR" Error Sequencing Error
PCR-based 0.085 0.277 3.36e-7
Culture-based 0.369 2.77e-5 4.86e-6
  • All of this analysis can be found on genemapster in </media/6TB_slot3/cjwei/Fate_Mapping/Non-Capture_Analysis/seq_error_[culture/PCR]> and is summarized in my 5/18 lab meeting presentation
    • The above calculates suggest that, while the culture-based method has a very low "PCR" error rate (as expected because bacteria should replicate inserts with very high fidelity), it also exhibited much higher Phi29 error.
  • We're not sure exactly what caused these vastly different error rates for phi29 slippage, so we'll be running a bunch of different tests this week to figure out what's going on.

Procedure

RCA

                       1x     3.5x
Template (1/10d)       5ul    -- <- used samples 1-3 (2nt, 3nt, 4nt)
RCA Primer (100uM)     2.5    8.75
dNTP                   0.8    2.8
10x Buff               2      7
Phi29                  1      3.5
Water                  8.7    30.45
-----------------------------------
                       20ul
  • Thermocycler: 37C 3hr -> 65C 10min -> 10C hold
  • I then performed ETOH precipitation and eluted into 20ul. These RCA reactions will then be aliquoted and used for various tests in order to keep most things consistent.

Test 1: Culture-based Method

BsrGI Digestion

  • I'll be using only 1/5 of the purified RCA product for this test. Consequently, I used the following mastermix for each of the three reactions/samples:
                         1x
RCA product              2ul
10x Cutsmart             5
100uM RE_BsrGI Oligo     5 <- I used the Redesigned Oligo from 4/15
Water                    27
-------------------------------
                         39ul
  • Thermocycler: 94C 2min -> 37C 3min -> Add 1ul BsrGI-HF -> 37C 2hr -> 80C 20min
  • I then purified this digestion product using ETOH purification and eluted into 22ul and saved 2ul of that for gel quantification

Second Strand Synthesis

  • After BsrGI digestion (and ETOH precipitation, eluted in 20ul water), we did second strand synthesis with the addition of the AmpR6.3 sequence (along with an 8N UMI2 replacing the index).
  • The following is the mastermix fo rthe second strand synthesis reaction:
                  1x
2nd UMI Oligo     5ul <- I used v4 of the 2nd UMI Oligo from 2/26/2016
KAPA HiFi         15
Template          10
  • We did two reactions per sample, using up all of the BsrGI product. After second strand synthesis, we combined both reactions, did ETOH precipitation, and eluted in 15ul.

De-phosphorylation with rSAP

  • After second strand synthesis, we must remove the phosphate group on the 5' end, which was left after BsrGI digestion. The leftover 5' phosphate group would prevent the blunt-end PCR product from ligating into the Zero Blunt TOPO plasmid.
  • Below is the mastermix for the rSAP dephosphorylation:
                 1x
Template         15ul
10x Cutsmart     2
rSAP             1
Water            2
  • Thermocycler: 37C 30min -> 65C 5min -> 10C forever.
  • We will do size selection of the second strand synthesis product and perform ETOH precipitation and elute in 6ul water (4ul is added to the TOPO reaction)

TOPO Blunt-end Ligation and Transformation

  • We will be doing four transformations (three samples + 1 positive control using the previous PCR of the given control insert)
rxn     Sample     Water     Salt     TOPO
1 (2nt) 4ul        0ul       1ul      1ul
2 (3nt) 4          0         1        1
3 (4nt) 4          0         1        1
4       (1/10d) 1  3         1        1 <- By using a 1/10d of the control insert, we can mimic 1ng of input into the TOPO reaction
Add DNA (10pg-100ng) and mix gently (do not pipette up/down)
                           |
                           V
                       30min Ice
                           |
                           V
                       42C 30sec
                           |
                           V
                       2min Ice <- I wasn't as stringent before on this waiting step.  So will make sure to wait 2min prior to adding SOC in order to allow bacteria to take up plasmids
                           |
                           V
    Add 250ul SOC and shake @37C for 3hr at 300rpm
                           |
                           V
                 Spread onto plates

Wash LB Agar+Kanamycin Plates

  • I washed the plates using the same procedure as outlined in <http://www.boneandcancer.org/protocolsa13.htm>
  • In summary, I added 4ml LB+Kanamycin broth to each plate and allowed solution to sit for a few minutes. Afterwards, I shook the plates in order to release the colonies into solution.
    • I tried pipetting directly on some colonies that were stuck in order to release.
    • Some agar was transferred into the solution because sometimes the p1000 pipette would accidentally scrape the gel.
  • I then proceeded with miniprep as per instructions with only 3 tubes (combined all plates belonging to the same reaction together)

EcoRI/BtsaI Double Digestion

                 rxn1     rxn2     rxn3
EcoRI-HF         1ul      1        1
DNA              20       39       40
10x Cutsmart     5        5        5
Water            29       10       9
  • Incubate: 37C 2hrs -> Add 1ul BtsaI (remove 2.5ul to run gel checking EcoRI digestion) -> 55C Overnight -> 65C 20min (deactivate EcoRI) -> 10C forever

Test 2: PCR-based Method

Test 3: phi29 Second Strand Synthesis, Ligation, and Direct Sequencing

Test 4: Sanger Sequencing Approach