Chris:LabNotes/FateMapping/Calendar/2016/2016-5-18
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Phi29 Slippage Rate Experiment - Method Comparison
Background
- Previously, I ran a PCR-based method and a culture-based method to calculate the phi29 slippage error rate. See <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-12-11> for PCR-based method and <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2016/2016-5-4> for culture-based method.
- Based on these two approaches, we get very disparate answers for the various slippage rates as summarized in the table below:
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
- We used the same exactly circularized oligos from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2016/2016-4-11>. Please note that these circularized products feature the 2016_02_26 version 4 oligos, which contain the full AmpF6.4 sequence in the bridge.
- I then wanted to run RCA on these circularized templates using the same mastermix as before (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2016/2016-4-21>):
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
- Incubate at RT for 30min -> Put on ice
- We added 2ul of each TOPO reaction to different vials of Top10 chemically competent cells (mix gently without pipetting up/down). In order to do transformation, we used the optimized protocol as on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2016/2016-4-13>
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
- I ran a similar double digestion reaction as on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2016/2016-4-28> but adjusted some of the input volumes in order to compensate for the decrease amount of plasmids extracted from colony harvesting
- Below are the double digestion reactions:
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