MM - Breed Me Deep Sir.mp4
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What can I say, we have a serious weakness for a tight small pumpkin shaped ass! It laterally makes us weak in the knees. Mac has a perfect pump-kin butt, truth be told, we invited him over for a solo stroke video and it quickly turned into more (cock sucking ass eating and facials, all on MMDirects) Butt as soon as we saw that pumpkin we knew we had to have him back for a full on fuck flick. When we see a sweet little pumpkin butt like Macs, it makes us crazy. As soon as we met Mack for the first video, we realized he was going to be fun, he is so sexy cute, has a great sense of humor and he was so comfortable with us and then when we saw that PHAT ASS we fell in lust lol when the video was over I put my camera down and jumped in the shower with Hunter, and much to our surprise Mack picked up the camera when we were out of the room and recorded us a little message telling us how much fun he had, so sexy and so sweet lol. You will love this deep ass fuck session
We present a simple CRISPR-Cas9 method to generate zebrafish F0 knockouts suitable for studying behaviour and other continuous traits. The protocol uses a set of three synthetic gRNAs per gene, combining multi-locus targeting with high mutagenesis at each locus. The method consistently converts > 90% of injected embryos into biallelic knockouts that show fully penetrant pigmentation phenotypes and near complete absence of wild-type alleles in deep sequencing data. In parallel, we developed a quick and cheap PCR-based tool to validate gRNAs whatever the nature of the mutant alleles. The F0 knockout protocol is easily adapted to generate biallelic mutations in up to three genes in individual animals. The populations of F0 knockout animals generated by the method are suitable for quantitative analysis of complex phenotypes, as demonstrated by mutation of a circadian clock component and by meticulous replication of multi-parameter behavioural phenotypes of a genetic model of epilepsy.
(A) The headloop score (HL score) for a given sample (here, tyr locus A larva 1) was calculated as the ratio between the headloop PCR band intensity (HL) and the standard PCR band intensity (std). As the standard PCR band intensity represents the sum of wild-type and mutated alleles, and the headloop PCR band intensity represents the mutated alleles only, the ratio approximates the proportion of mutated alleles in the sample. (B) Percentage of mutated reads (deep sequencing) as a function of headloop score. Each data point corresponds to one targeted locus in one F0 knockout animal. Some samples were artificially created to simulate mediocre gRNAs by mixing genomic DNA from an injected embryo with genomic DNA from the uninjected control in a 1:1 (diluted , squares) or 1:3 ratio (diluted , triangles). For these, the percentage of mutated reads was not measured by deep sequencing but estimated by dividing the percentage of mutated reads of the original sample by 2 (diluted ) or 4 (diluted ). The dark grey dashed line is the line of best fit by linear regression: proportion of mutated reads = 0.33 + 0.69 headloop score; R2 = 0.44. Headloop score is a significant predictor of the proportion of mutated reads, p
In the first round, each gRNA set is injected followed by deep sequencing or headloop PCR to confirm mutagenesis, thereby controlling the false negative rate of a screen. Headloop PCR is cheap, robust, and requires only a single step, which makes it easily adapted to high-throughput screening. No specialist equipment is required, as opposed to qPCR (Yu et al., 2014), high resolution melting analysis (HRMA) (Samarut et al., 2016), or fluorescent PCR (Carrington et al., 2015). Unlike deep sequencing, qPCR, and HRMA, it is also flexible with respect to the size of amplicons and so is sensitive to a wide range of alleles, from small indels to large deletions between targeted loci. It can be used to assay the efficiency of any gRNA, with no restrictions on target sequence that might be imposed by the use of restriction fragment length polymorphism (Jao et al., 2013), for example. The products of headloop PCR are also compatible with different sequencing methods, should further analysis of mutant haplotypes be required.
It is precisely because discovering novel phenotypes is the main challenge of F0 knockout methods that an essential criterion for success was achieving high phenotypic penetrance and close to complete removal of wild-type alleles for test genes. When studying phenotypes that vary continuously in the population, such as locomotor activity (Figure 6) or circadian clock period (Figure 5B), it is not possible to readily identify animals in the F0 knockout population that display a phenotype, in contrast to situations in which animals exhibit overt morphological differences. Therefore, the majority of the animals in the F0 knockout population need to be bona fide knockouts. Continuous traits often have the added challenge that they are not phenotypes whose spatial variation is evident in individual animals (Watson et al., 2020), in contrast to developmental phenotypes like formation of electrical synapses (Shah et al., 2015) or distribution of microglia (Kuil et al., 2019). Therefore, most cells in each F0 knockout animal must carry biallelic loss-of-function mutations. With the pigmentation phenotypes golden (slc24a5 knockout) and sandy (tyr knockout), and the deep sequencing data at 32 targeted sites, we demonstrate that the F0 knockouts generated by the present method show very little mosaicism, in the sense that most wild-type alleles are removed, and hence can be used to discover novel phenotypes that require most or all the cells of most F0 knockout animals to be mutated.
In the case of the small indels around each targeted site, we discovered through deep sequencing that there was substantial diversity in alleles, including within individual animals. Most mutations at each locus are present at low frequencies in individual animals (> 95% mutations detected at each locus are present at frequencies below 10% in individual animals, Author response image 1). This may be because the mutation happened later in development or in a cell that did not contribute many daughter cells. High coverage is necessary to detect and quantify this diversity. Many of these alleles would likely be missed by the lower coverage of an amplification-free experiment.
To answer specifically the first question raised, the order when targeting gradually more loci (Figure 1E,F) followed the IDT ranking when selecting a single crRNA per exon (see Material and methods, section RNP pooling). Therefore, when targeting a single locus, only the top predicted gRNA is injected; when targeting two loci, the top two predicted gRNAs are injected together; etc. Based on the deep sequencing data (Figure 2A), this amounts to essentially the same as picking the order randomly within the top 3 or 4 gRNAs, as the top predicted gRNA of the set is only rarely the most mutagenic one in practice. Indeed, when we sequenced 10 genes targeted by three or four-gRNA sets, there were only 3 sets in which the top predicted gRNA by IDT (gRNA A) was also the top performing gRNA (slc24a5, tbx5a, scn1lab). This is what is expected by chance alone (if the most mutagenic gRNA of each set was drawn randomly, 31% of gRNA sets would have the top predicted gRNA as most mutagenic gRNA). In other words, it is not possible to guess above chance the IDT ranking from the mutagenesis levels alone. 59ce067264
https://www.samposd.com/forum/ilban/plants-vs-zombies-battle-for-neighborville-fre
MMOexp-Lineage 2M: PvP Meets PvE—How to Win Raids Against Rival Clans
Lineage 2M offers players one of the most expansive MMORPG experiences on mobile and PC, combining large-scale battles, dynamic PvP, and cooperative PvE encounters. Among its many features, raids stand out as some of the most challenging and rewarding content in the game. Whether you’re facing off against massive world bosses or organizing clan-based raid events L2M Diamonds, success depends on preparation, teamwork, and the right strategy.
At MMOexp, we know how difficult raid progression can be—especially when competing against other clans or farming for rare loot. That’s why we’ve put together this comprehensive guide to help you and your team master Lineage 2M raids. From preparation tips and team composition to combat tactics and rewards, this guide will ensure that your group has the knowledge to dominate every raid encounter.
What Are Raids in Lineage 2M?
In Lineage 2M, raids are large-scale PvE encounters where players band together to take down powerful bosses. Unlike standard dungeons or elite zones, raid bosses feature:
High HP pools and devastating AoE attacks.
Unique mechanics requiring teamwork and coordination.
Timed events where multiple clans compete for kills.
Exclusive loot drops including rare weapons, armor, and crafting materials.
Raids are not just battles—they are community-driven events that bring clans together, spark competition, and fuel server-wide rivalries.
Why Raids Matter
Raids are more than just exciting battles; they’re a crucial part of Lineage 2M’s progression system.
1. Rare Loot
Raid bosses often drop legendary items, high-tier crafting materials, and rare consumables that are nearly impossible to acquire elsewhere.
2. Clan Prestige
Defeating raid bosses—especially in contested fights—brings prestige to your clan. Owning raid victories helps establish dominance on your server.
3. Adena and Resources
Raids yield large amounts of Adena, experience, and valuable resources for both individual players and entire clans.
4. PvP Opportunities
Many raid zones are open-world PvP arenas, where clans fight both the boss and each other. Victory often depends on managing both enemies and rivals.
Preparing for Raids
Success in raids begins long before the battle starts. Preparation ensures your team isn’t caught off guard by mechanics or rival clans.
1. Gear Up Properly
Weapons: Choose gear that matches your class role. DPS players need damage-focused weapons, while tanks prioritize survivability.
Armor: High-defense and resistance gear is essential for mitigating raid boss AoE damage.
Consumables: Stock up on healing potions, buffs, scrolls, and resurrection items.
Pro Tip: If farming gear is too time-consuming, you can always buy Lineage 2M weapons, armor, and consumables at MMOexp for an affordable and reliable advantage.
2. Assemble a Balanced Team
A strong raid group requires class diversity. Balance ensures survival and efficiency:
Tanks to absorb boss damage.
Healers to sustain the team.
DPS (Melee & Ranged) to maximize damage.
Support classes to provide buffs, debuffs, and crowd control.
3. Scout the Boss
Before engaging, study the raid boss’s attack patterns, enrage timers, and mechanics. Many bosses punish disorganized groups with devastating AoEs or summoning minions.
4. Timing and Coordination
Raids often attract rival clans. Organizing your raid schedule and arriving early ensures you’re prepared to secure the boss before others.
Team Composition Strategies
Tanks – The Frontline Heroes
Tanks are responsible for holding aggro and protecting the party from boss attacks. Key strategies include:
Maintaining positioning so AoE attacks don’t hit the entire group.
Rotating defensive cooldowns to survive boss bursts.
Communicating with healers to anticipate heavy damage phases.
DPS – The Damage Dealers
Your DPS are the engine of the raid, pushing through the boss’s massive health pool. Strategies:
Focus on target priority (boss first, minions second if required).
Avoid unnecessary damage by dodging AoEs.
Use cooldowns during enrage phases for maximum efficiency.
Healers – The Lifeline of the Raid
Healers keep the group alive and must react quickly:
Prioritize tanks while maintaining party-wide healing.
Use mana-efficient skills to avoid running dry mid-battle.
Pre-cast heals during predictable boss AoEs.
Support – The Unsung Heroes
Supports make raids significantly easier:
Apply buffs to increase DPS and survivability.
Debuff the boss to reduce incoming damage.
Control adds with crowd-control skills when necessary.
Raid Combat Tactics
Positioning Is Key – Spread out to minimize AoE damage and avoid chain effects.
Focus Fire – All DPS should focus on the same target for faster kill times.
Manage Adds – Many bosses summon adds. Assign an off-tank or DPS group to handle them.
Interrupt Skills – Some bosses cast devastating abilities that can be interrupted. Assign players to monitor and cancel these casts.
Survive the Enrage – Most raid bosses have enrage timers. If you don’t kill them in time, they’ll wipe the raid. Maximize DPS during burn phases.
Dealing with Rival Clans
Raids in Lineage 2M often double as PvP battlefields. Your raid group must be prepared for both the boss and competing clans.
Tips for Winning PvP During Raids:
Assign a portion of your DPS to focus on rival players.
Tanks can disrupt enemies by taunting and stunning their healers.
Control the raid zone early to prevent rivals from interrupting your fight.
Use voice communication to coordinate PvP and PvE simultaneously.
Common Raid Boss Mechanics
Each raid boss in Lineage 2M is unique, but many share common mechanics:
Area Denial: Bosses drop damaging zones that force players to reposition.
Add Waves: Summoned enemies distract and overwhelm teams.
Debuff Stacking: Bosses apply stacking debuffs that tanks must manage.
One-Shot Mechanics: High-damage skills must be avoided or mitigated.
Knowing these mechanics ahead of time makes your raid significantly easier.
Rewards & Progression
Raids reward both individuals and clans with some of the best loot in Lineage 2M.
Weapons & Armor: Epic and legendary-tier gear.
Crafting Materials: Essential for high-level gear upgrades.
Adena: Large sums that support future progression.
Clan Rewards: Victories strengthen your clan’s reputation and unlock benefits.
Pro Tip: If you miss out on certain drops, MMOexp provides a reliable way to purchase rare gear directly—ensuring you stay competitive even if RNG isn’t on your side.
MMOexp Tips for Raid Success
Communicate Constantly – Use voice chat for real-time coordination.
Assign Roles Clearly – Everyone should know their job before the battle starts.
Arrive Early – Secure raid zones before rival clans arrive.
Adapt Quickly – If a strategy isn’t working, switch tactics mid-fight.
Keep Improving Gear – Stronger weapons and armor always make raids easier.
MMOexp – Your Partner in Raid Progression
While skill and strategy are critical in raids, gear remains the foundation of success. MMOexp helps you stay ahead by offering:
Affordable Lineage 2M weapons, armor, and items.
Fast, safe transactions with guaranteed delivery.
A wide selection of gear for every class and role.
24/7 support for players around the globe.
With MMOexp, you can spend less time grinding and more time mastering raid strategies with your team.
Conclusion
Raids in Lineage 2M are the ultimate test of teamwork, strategy, and preparation. They demand well-coordinated groups, smart class composition, and the ability to adapt quickly under pressure. By following MMOexp’s raid strategies—balancing roles buy L2M Diamonds, mastering boss mechanics, and managing rival clans—you can lead your team to victory and claim the rare rewards that make raids so valuable.
And remember: while teamwork and skill win battles, the right gear wins wars. With MMOexp’s affordable and reliable marketplace, you’ll always have the weapons, armor, and resources needed to face any raid boss with confidence.
Gear up, rally your clan, and conquer the raids of Aden—victory awaits.